Swee Lay Thein: Good morning, everybody,
I think we are running a bit late,
so we must go and get started as soon as we can.
I really like to extend a huge welcome
to all of you on behalf of the organizing committee,
which is myself, Monika, Jennifer, and John Tisdale
to the 17th Sickle Cell in focus meeting.
I am so delighted to see so many in person,
but in fact more than 500 actually registered.
And they will be listening online,
and that is the reason why we are a bit late,
because we want to make sure that these guys
who've registered can actually tune in.
So, the housekeeping things will come in a bit later,
because I think we should get started ASAP.
And with that I will like to introduce,
Professor Minerva Thame,
who is the dean of the medical school in Jamaica and --
Is she coming in person?
[laughter]
Swee Lay Thein: Oh, Welcome. Would you like to come and talk?
Thank you so much, I didn't realize you were there waiting.
Minerva Thame: Thank you, very much.
Swee Lay Thein: Thank you for coming.
Minerva Thame: Yes, I'm here in person.
I think the last time we were virtual, right?
The last meeting, so here is my opportunity
to get to see all of these faces.
So, chair, delegates from all over the world,
colleagues, good morning.
Thank you for inviting me to bring greetings
at the 17th Annual Sickle Cell in Focus Conference.
I am delighted that the National Heart, Lung,
and Blood Institute is cohosting the 24th Annual Conference
for the University of the West Indies
once again. Such collaboration
helps to build and strengthen institutions
as they share in scholarship.
I want to also recognize the powerful speakers
we have on this agenda
to present from both Institutions
who no doubt will give us a treat over the next two days.
Sickle Cell Disease
is such an important disease for us here in Jamaica.
One in 300 babies are born in Jamaica with homozygous SS,
while one in 500 are born with a C disease.
Ten percent of our population carries a sickle cell trait.
With the figures, the burden of disease in Jamaica,
is quite high.
Fortunately for us, sickle cell disease
has been extensively studied in Jamacia,
and these studies have informed
and led to improve management of our children
with sickle cell disease.
And has improved significantly the rate of survival.
Many of our children use to die very young.
But due to research conducted,
which have led to the implementation of policies,
the lifespan has improved significantly.
So much has the life spanned improved that the survival
has now seen our children growing up to adulthood,
and into reproductive age.
This has therefore, allowed us to do more studies
on pregnant women with sickle cell disease,
which is has been able to add valuable knowledge
to the global literature.
Professor Graham Serjeant, who is dear to my heart.
Who has mentored me for many years;
who I still work with,
has been a name that's synonymous
with sickle cell disease in Jamacia.
He was the first director of the sickle cell unit.
And following the work done on his long-life birth cohort.
He has added through his research,
knowledge which we now have.
Other persons have worked within the sickle cell unit,
and I will not call names, because I'd get into trouble.
But they have done extensive work,
but one of them which I must say I am very proud of is;
a newborn screening, which now is a reality in Jamacia.
And a very important initiative for our island.
We have so much more work to do as we strive
to serve this important group within our population.
This conference will continue to educate us
and from discussions, I am sure that new ideas
and research questions will emerge.
We will receive updates on,
screening for sickle cell disease,
complexity of common complications
in sickle cell disease,
such as pain management, sleep and reproductive health,
therapies for sickle cell disease,
both curative and noncurative,
and ongoing challenges in the management
of sickle cell complications.
I wish you all a very successful and fruitful conference.
And I know the participants will be exposed to rich information,
which will enhance our knowledge of sickle cell disease.
And we will serve this very important population.
Thank you very much.
[applause]
Swee Lay Thein: Thank you, Professor Thame.
So, we -- Dr. Childs, who is the director of initial VI
[phonetic sp], unfortunately, hoped to come,
he had to cancel at the last minute,
but he will give welcome remarks which we will now present.
I just hope this be ready [sic].
Okay, Thank you, Ric.
Richard Childs: -- Healthcare professionals
and advocates, my name is Dr. Richard Childs,
and I am the scientific director at the National Heart, Lung,
and Blood Institute or NHLBI
at the National Institutes of Health.
And it is my distinct pleasure to welcome all of you
to the 17th Annual Sickle Cell in Focus Conference,
co-hosted by both the NHLBI
and the University of the West Indies.
This year we're excited to gather both in person
in beautiful Kingston, Jamacia and virtually reflected
on the global importance of this event,
and the broad reach of our mission.
While we may not all be in the same physical space,
the urgency of our mission, and the impact of our work,
are just as powerful. Sickle cell disease
remains a significant public health challenge,
effecting over 100,000 U.S. citizens
and millions of patients world-wide.
It's a disease that brings complexities,
and inequities that we as a community
must continue to address.
This annual conference serves as a vital platform
to share the latest advances in scientific research,
in clinical care, and global health strategies,
as we work together to improve the lives of those living with
and suffering from sickle cell disease.
At the NHLBI, our commitment to sickle cell research
is unwavering.
We've made remarkable strides in both curative
and noncurative therapies,
while also focusing on innovative treatments
that address the diverse complications
associated with this disease.
Some of the key initiatives that we are advancing
include the cure sickle cell initiative.
Which is working to accelerate the development
of genetic therapies
aimed to find a way to cure sickle cell disease.
This bold endeavor holds the potential
to transform the future of treatment for this disorder,
and just this year has resulted in two new FDA
approved therapies using gene therapy.
Our focus on health disparities aims to ensure
that those disproportionately affected by sickle cell disease,
particular in
the African-American and minority communities,
receive equitable care, and opportunities to participate
in cutting edge research and clinical trials.
This year's Sickle Cell in Focus Conference
will address several critical and important themes,
including updates on screening for sickle cell disease,
pain management, sleep, and reproductive health,
and the complexities of curative and noncurative therapies.
We will also delve into ongoing challenges
that many patients face in managing this condition,
including complications such as stroke, organ damage,
and chronic pain.
Importantly, the voices of the patient's and clinicians
will shape much of the dialogue of this meeting.
Allowing us to collectively explore new ways
to approach treatment and care. The agenda in this meeting
is packed with world-class educational content
featuring renowned experts, who will share their insights
on both the latest scientific development
and real-world patient experiences.
Our sessions will include interactive panels,
where clinicians, researchers, and patients will come together
to discuss the future of sickle cell management and treatment,
focusing on both cutting edge innovations,
and the everyday challenges
that are associated with this disorder.
We are especially grateful for the partnership
between the NHLBI and the University of West Indies
and the Sickle Cell Unit.
This collaboration which continues to grow and thrive,
underscores the global nature of our work.
Particularly in regions where sickle cell disease is endemic.
Together we can address not only the scientific
and clinical aspects of this disease,
but also the social and systemic barriers to its care.
In closing, I want to emphasis
the importance of this conference,
not just as a forum for scientific exchange,
but as a source of hope for the sickle cell community.
It's through events like these where we come together,
share knowledge, and build collaborations,
that we make tangible progress toward a world
where sickle cell disease will no longer impose
the significant burden that it does today.
I want to thank all of you for your attendance of this meeting,
your dedication to the cause,
and I look forward to the engaging discussions,
and the invaluable insights that will ensue from this conference.
Let's make the next two days, impactful, innovative,
and inspiring for the future of sickle cell disease
care and research.
I wish all of you a great conference.
Swee Lay Thein: Can you hear me? Yes, I can hear myself now.
[laughs] Good morning, every one,
and my own very warm welcome to beautiful Jamacia,
please enjoy the scenery, in the breaks,
not right now [laughs]. [laughter]
Swee Lay Thein: I really want to thank our two leaders,
Minerva Thame the head of her school,
head of her faculty of medical sciences
and Ric Child from NHLBI
for the inspiring, very hopeful words,
and words of collaboration and engagement
in moving the field forward.
Sorry for the delay, so we will get straight into session one.
And to start us off --
by the way if you do the QR code,
it will take you straight to the booklet, okay,
with the whole agenda as well as detailed bios of our speakers.
So please take a moment to do that
and the QR codes are all around the room,
and on your tables probably.
We'll go right into our first speaker.
Welcoming, Dr. Daniel Dexter,
who is training in pediatric hematology.
And is currently a hematology registrar
at King's College hospital, London, UK.
He will speak to us on point of care testing,
the promise and the rule. Welcome, Daniel.
Daniel Dexter: Thank you everybody.
Female Speaker: [inaudible] King's College [inaudible]
Daniel Dexter: Yes, sorry, just started.
Female Speaker: [inaudible] [laughter]
Daniel Dexter: Oh, okay. Is there a clicker?
Thank you, very much.
Female Speaker: [inaudible] point.
Male Speaker: To the back
Female Speaker: To the back
Daniel Dexter: Okay, technical issues,
we'll learn from my mistakes. I just wanted to start by
thanking the organizers for inviting me here.
It's a really great honor to speak in front of all of you.
I follow many of your research, throughout my career.
I have been invited to -- my name's Dan,
and I'm a hematology registrar at King's in London.
And I have been invited to speak about exciting new developments
in point of care testing for sickle cell disease.
Oh, it's working, so the global prevalence
of sickle cell disorders is growing.
Last year in the [unintelligible]
the global burden of disease study,
which drew upon all available national epidemiological data
for sickle cell determined
that upwards of half a million infants
will be born every year with sickle cell disorder.
Estimates do vary, and Fred Peal's [phonetic sp]
work estimates around 300,000 to 350,000 infants
will be born with sickle cell anemia each year.
Yet presently most infants born with a sickle cell disorder
will not obtain a formal diagnosis.
Access to diagnosis is most scarce,
where the burden of disease is greatest.
This is also true of access to care.
As we know, the majority of sickle cell disease
affected births occur in Sub-Saharan Africa
where health resource scarcity is most acute.
This is reflected in profound inequities of disease outcomes.
The true extent of sickle cell's
impact on individuals in societies
is hidden with in global health statistics.
The map on the left of the screen from the GBD,
oh, I've gone backwards, sorry,
GBD study illustrates the significant discrepancy
of available data for SCD by country.
Data capture is really lucky in remote rural settings,
especial in Africa, much more granular in detail
than better resourced nations.
Sickle cell is comparative neglected disease for funding,
when you think about the funding for cystic fibrosis
in high income countries or HIV in low-income settings.
What we do know is that sickle cell is a major
and underestimated cause of early childhood mortality,
and it places a huge burden on health resources
in low-income settings.
The demand for screening has never been greater.
Oops, sorry, did you click? Oop.
So why do we want to screen?
Well, early diagnosis acts as the gateway
to timely and lifesaving interventions.
The seminole [phonetic sp]
Jamaican Cohort Study, as was mentioned earlier,
demonstrated the impact of early diagnosis
when twinned to low-cost interventions
such as parental counselling and [unintelligible]
prophylaxes in achieving a reduction
in childhood mortality.
In recent decades, we have seen the development
of national newborn screening programs
across North America, Europe and also in Brazil.
These sizable linguistical endeavors,
benefit from economies of scale, and timely identification,
and enrollment of positive infants into comprehensive care,
has transformed the prognosis of infants
born with the disease.
In the UK, sickle cell is the most common test
that we screen for on the newborn screening program.
Effecting roughly one and two and a half thousand births.
Ninety-nine percent of our children in the UK
with sickle cell disease reach adulthood.
Just last year, France has moved from a targeted
to a universal screening approach.
We have the tools, to not just impact mortality,
but also support a better life
for those born with sickle cell everywhere.
Comprehensive care goes beyond prophylaxes
and reactive measures.
Hydroxyurea, so you say urea, we say carbalites
[phonetic sp]
continues to demonstrate the ability
to effectively modify disease,
reduce complications, and improve prognosis.
Ground breaking studies, such as the Reach Studies,
conducted in Africa,
demonstrated that it's safe and effective in limited resource
settings with malaria aminesity [phonetic sp].
This includes using the maximum dosing strategy,
which achieved best outcomes.
Yet as we enter the era of gene therapy,
and cure, potentially;
diagnosis is still inaccessible to most,
which represents a tragic missed opportunity
to avoid countless preventable deaths.
Over the past 30 years,
pioneering newborn screening programs
in the Caribbean and Africa
have made significant achievements
in demonstrating the feasibility of screening.
Kumasi, the garden city in Ghana,
was the first African country to establish a pilot program.
Most pilots operate using a centralized laboratory approach,
which is reliant on laboratory-based techniques,
but these programs have supported to development
of national reference laboratories
and epidemiological data registries.
And they have also been able to link diagnosis
to comprehensive care.
But what has prevented the scale up of this pilot programs?
It's to do with the challenges inherent
with the centralized screening approach.
Laboratory best techniques can be costly,
they require highly trained personnel,
and in the experience of the Uganda pilot,
that was the most costly factor of all,
steady supply of re-agency consumables,
and access to technical support when they break down.
Also, there is significant linguistical barriers.
How do you reach all of the infants that you need to screen,
especially in rural settings?
How do you maintain these extensive sample
transport networks?
Also, when diagnosis is not at the point of care,
there is a long turn around time.
How do you contact trace your positive infants?
It can be hard sometimes to reach rural addresses,
and this has led to a significant loss to follow up.
Other barriers include data management,
the ethical obligation to link diagnosis to care,
and not be underestimated,
the acceptability of screening to communities.
In Uganda they screen each child for $4.67,
but the turn around time is around 16 days.
And the Angolan experience is around
just over 50 percent of their screened positive infants
were actually enrolled in comprehensive care.
So, there's a big missed opportunity.
Just a brief overview
of the conventional screening diagnostics.
They are mostly electric phoretic techniques,
but you do have notable mentions for high-output fancy
things like Tandem Mass Spectrometry,
and a greater role for DNA sequencing.
I'll briefly touch upon the two most commonly used ones,
which are HPLC,
which is a qualitive as well as a quantitive assay.
It requires highly trained operators;
it's rapid in high output.
It can help rationalize ambiguous results,
by other methods.
But it requires availability of sophisticated technical supports
and lots of consumables. It's relatively expensive.
By contrast isoelectric
focusing which you can see here with the nice bands,
is a semiautomated mostly qualitive measure.
We can run lots of large volumes,
and it's cheaper than HPLC. And this is the modality
that many of the pilot programs use in Africa.
So why are we excited about point of care testing?
For over a decade now these point-of-care test
have been highly impactful in HIV and malaria programming.
They provide diagnosis at the point of need,
and they essentially eliminate turnaround times,
and impact loss to follow up.
They are ideally suited to the primary care setting.
And I know there is lots of movement now
looking to see if sickle cell screening
can be optimized for the immunization clinic setting,
much less busy than the crazy environment
of a post-natal ward.
And there is a very high uptake of immunization in countries
with a high sickle cell burden.
Also, the persistence of fetal hemoglobin permits
an extended window for screening of sickle cell
compared to other congenital metabolic disorders,
which need treatment in the first weeks of life.
Point of care test tend to be user friendly, portable,
and people are familiar with them,
think pregnancy test, now COVID test.
Affordability is a big big factor here.
Now, financial mechanism
such as the global funds pool procurement mechanism,
we have a single buyer, a tough negotiate --
they can negotiate hard with companies to get very low cost
for the HIV/malaria test, down to 30 cents a test.
And then order millions of the tests on sustainable supply,
which they distribute to their partners.
This is the kind of thing
which can make testing more affordable.
Just a brief nod to "this old chestnut."
[laughter]
Daniel Dexter: Sickle cell solubility test still in use,
not confined to history, just to say,
it's not appropriate for newborn screening,
because it's inaccurate in large amounts of hemoglobin F,
and doesn't differentiate between sickle and trait,
but it's still widely used.
So, just to talk about some sickle cell specific challenges
that point of care test manufacturers
and designers need to overcome.
It's really important that any test can differentiate
between disease and trait.
The tests need to be accurate in samples
with high amounts of hemoglobin F. Ninety percent
of a fetal blood sample might constitute hemoglobin F.
The need to have low limits of detection
for the hemoglobin variance they are looking for.
So, at least under 10 percent for hemoglobins S, C, and A,
ideally, around 4-5 percent.
We should praise the ability of test
to diagnosis compound heterozygotes
or at least recognize the limitations
if they cannot do that,
as I will talk about in a couple of slides.
Also, in transfused patients
if you have very low limits of detection of HbA,
and you're a transfused patient with sickle cell disease.
And you have one of these tests,
they might test as having trait rather than disease.
So, lateral flow immunoassays: this is an antibody-based format
which has been the workhorse
of point of care modalities for years.
The qualities of these antibodies
is essential to determining the accuracy of the test [sic].
The use of monoclonal antibodies
tends to lead to a more specific test.
Also in the manufacture process,
the amount of antibody in the device
is very important to the test performance.
They can be competitive or sandwich formats,
which will affect how they are read in terms of their results.
But they are self-contained, disposable, cheap, and familiar.
They require no electricity,
and we have two well established test
for sickle cell disease with [unintelligible]
in this format.
And as I will talk about, there is a tidal wave of test
coming from India in the same format.
So, in the process of updating my previous paper on this topic,
I was looking at the --
the Indian government announced last year
that they want to try an eliminate sickle cell disease.
India has the third highest incident
of sickle cell births every year.
They want to screen 17 million people in three years,
mostly in primary care.
And Indian industry has responded by producing
15 new point-of-care tests in the last year alone,
which have all been validated by the government.
They're commercially available.
Now, I got sent this document
from the Indian Council of Medical Research,
and my eyeballs almost fell out of my head.
Because I thought that we only had
two actual lateral flow tests, but there are so many now.
I must say that the validation processes are quite opaque,
and I haven't been able to find
any peer review of the validation processes.
I think there has been some [unintelligible]
report that some of the tests aren't performing
as well in the field as well as the validation data suggest.
So, the Indian FDA,
the CDSCO has reclassified sickle cell diagnostics
to a more stringent class;
I think to get some oversight on this.
However, this kind of industry activity
might lead to a lowered cost of tests in the future.
So, just a quick run through of HemoType and SickleSCAN.
HemoType is a competitive lateral flow test
which means the result is read,
a positive result is discerned by the absence of a line.
It's performed in five steps.
It's a monoclonal based test, no buffers required.
You put some blood into some water into a test tube,
insert the test strip, wait for ten minutes,
don't undercook it or overcook it,
because it can affect the reading,
and it gives you result.
The Sickle SCAN test is a very neat test in a cartridge format.
It is a sandwich type polyclonal test,
polyclonal antibodies. It does require a buffer,
but that's applied in the pack and it runs in five minutes.
This is just a screen, just a graph,
just to show you some basic technical details of the test.
I've included an Indian test just out of interest.
It was a rather arbitrary choice.
SickleCheck were the only company
that responded to my deluge of emails,
I was trying to see what's out there.
So, they detect hemoglobin A, S, and C,
but not in the case of SickleCheck,
which only has A and S,
so, its geographical relevance is less.
They have low limits of detection.
The cost is the crucial factor here
and HemoType is the cheapest, retailing for $1.49.
They are stable at room temperature for up to two years.
Field validation: these are accurate test,
and there were a number of studies
with HemoType and SickleSCAN in peer review literature,
of course many settings in Asia, and Africa, and the Caribbean.
I won't talk about the most exciting studies,
that I'll leave to other speakers today.
But they have been validated in newborn screening frame work
as effective tools, which is very exciting.
Operational issues: There was some concern
about the counterintuitive readings
of a HemoType test, but the papers,
and the experience of users with actual training
and retraining of healthcare workers,
these issues can be ironed out.
The SickleSCAN has a relatively weak HbA band,
but in general the experience with these tests
are effective and impactful [sic].
One more thing about HemoType
is that you can use it not just with whole blood,
but with debiased samples too, try blood spots.
Now for something completely different,
the Hemex Gazelle is a completely different class
of point-of-care-test.
It can be described as a portable laboratory of sorts,
an electro phoresies in a box.
It relies on a disposable cartridge,
which you insert into a box
which retails in Africa for $800.
The cartridges cost $2.00 to $2.50;
within the cartridge you have a cellulose acid
gel electro phoresies with two electrodes either end.
You put it into the box and apply the blood sample,
and as the electrical field is applied
and the hemoglobins migrate,
and image acquisition system takes lots of pictures.
Which is read by a very sophisticated software,
and the software is the crucial thing here,
because it's permitted expanding capability of the device
with different iterations of it.
It detects not only A, S, and C, but hemoglobin F, E, and A2.
The results can be uploaded to a cloud application
which is good for data management.
And they have neat printout which can be emailed to patients
or saved on your computer.
Its battery run or plugged into electrical means.
It takes one sample; it can run one sample every 10 minutes.
It is also multi-model,
and the company provide cartridges
which can diagnosis malaria,
and they are looking in to developing tests
for full blood count too.
The device is said to able to last around 10,000 tests,
so 3 to 5 years.
It's performed well in the field.
The initial iteration was not suitable for newborn screening,
because the limit of detection for hemoglobin S was too high.
But a software update
means now the limits of detection is 4 percent,
and it is validated and accurate for newborn screening
for sickle cell disease. There was some issues I think,
with some normal variant hemoglobin
being misdiagnosed as trait,
but that just confirms why we need a different methodology
as a confirmatory test [sic].
This test also enables beta thalassemia diagnosis
because a software update enables
hemoglobin A2 quantification.
So, it's quite a powerful potential in this device.
And this device is too been validated
in a newborn screening framework in Ghana.
Oh wait, have we been there already?
Oh, sorry, I think, I've got an extra slide here.
One last thing, you can use this device
for hemoglobin F quantification.
It has been used in this regard in Ghana
as well as Epostraeha [phonetic sp].
This means you can use it
for sophisticated Hydroxyurea monitoring.
Can't we get our patients up?
We can really monitor how effective treatments
is going for them [sic].
There is a brief slide on licensing.
They're licensed across much of Africa, Asia, and Europe.
I just wanted to mention the reassured criteria.
These are the WHO checklist really to help us access
what the suitability of new point-of-care tests
following com settings as they come online.
And I think, without laboring the point,
the tests that I have discussed today,
do fulfill most of these criteria.
However, questions remain as to the impact
that these tests will have. Am I on the right screen?
I am. The affordable tests threshold
is likely to vary country to country.
Currently, the tests cost $1.50 to $2.50 each,
but in reality, this is unlikely to reflect the true costs
when tariffs and distribution fees are factored in.
In which invariable raise costs by several orders.
In my opinion, we need to reduce the costs of these test
to achieve true affordability.
And we need to find ways of maintaining a steady supply
of them to get them into the hands of the people
that want to use them.
Reference laboratories with goal standing tests
remain important for confirmatory testing,
but also for developing lab infrastructure
and rationalizing ambiguous results.
There might come a point where lab-based methods
can make efficiencies in low-income countries
that help them achieve cost efficiency
through economies of scale.
Furthermore, we need to identify optimal strategies
for how we use these point-of-care tests.
As the market becomes more competitive,
there will be validation and regulatory issues
to ensure that only the effective and quality test
get licensed.
Other wide issued central to effective screening,
require addressing as well including,
how to manage and potentiate data,
ensuring diagnosis is linked to care,
and building acceptability of screening in communities.
Strategies to Reduce Cost: I am not sure
what's happened to that image there.
So, in effort to funding strategy
such as the pool procurement mechanism
would be very helpful here.
ASH, with their Cancer program are using a similar method
to buy up millions of point-of-care tests
which they then distribute
to their partner countries and labs.
It also guarantees a sustainable supply.
We need more government support,
not just for budgetary commitments
to sickle cell programs,
but also can we get them to waive taxes on resources
used in sickle cell care and screening?
To reduce the tariffs that we have to pay
to import these devices.
And maybe it's a bit pie in the sky,
but if we could manufacture point-of-care tests
and hydroxy carmide
[phonetic sp]in Africa and then coordinate their distribution
throughout the continent to reduce the cost.
So, in summary, we have available affective
point-of-care test.
They're commercially available; we can buy them.
They are affective; and they're accurate,
and people are using them.
The gazelle device is quite a sophisticated test,
which can add more than just screening, has clinical utility.
There are more products emerging,
and these point-of-care tests
are likely to be highly effective tools
that supports our efforts to increase access to diagnosis.
However, challenges still remain:
We need to achieve true affordability;
We need to ensure quality;
And there is still a very important role
for lab-based approaches as the goal standards.
Thank you very much. I hope I kept to time.
[applause]
Swee Lay Thein: Don't go, you might have questions.
Daniel Dexter: Oh, sorry, yeah.
Swee Lay Thein: [laughs] Thank you, so much, Dr. Dexter.
We'll open the floor for questions,
and as you get your questions together,
I'm also wanting to say, welcome to our zoom participants.
And please do go ahead and post your questions in zoom
as well if you have one.
Anything for Dr. Dexter. You were so clear.
Female Speaker: Can I ask one question?
Daniel Dexter: Sure.
Female Speaker: So, near the end from what you're telling me,
the Gazelle might be an [inaudible]
investment in the long term.
It cost a bit more to start with but then for the
[inaudible] that you can make.
Daniel Dexter: I think, it is very setting specific.
I think the capabilities of a device like Gazelle
might add so much to rural or rather remote locations
which might not have access to quantification for example.
It's still relatively low cost compared to other measures.
I think it also --
I know that some people are using HemoType
as a mass screening tool. I think, it comes down to cost
and what other resources are available in each setting.
But certainly, I think the multi-model dynamic
of this device is really useful.
Female Speaker: So, in the end, probably,
you might want to [unintelligible]
level screening which is cheap that can reach many people,
and then if there are any [unintelligible]
you could send [unintelligible]
maybe. Is that correct? It might be a good option.
Daniel Dexter: Yes, I think that's --
I think that probably speakers today
will talk about platforms exactly like this.
So, using the point-of-care tests
as the initial screening tool,
and then having confirmatory testing in a lab-based assay.
However, a mentor of mine in Uganda, is --
they are doing the complete opposite.
They're relying on IEF for screening,
and they use HemoType in their lab as a confirmatory test.
So, I think, when I talked about identifying optimal strategies,
I think, they're still being investigated,
but definitely point-of-care tests allow flexibility
for screening and diagnostic options
which is incredibly useful.
Male Speaker: Great review, thank you.
Could you talk a little bit more about the heterogeneity
of the different programs at least throughout Africa?
You alluded to it a little bit among different countries.
But I guess the other thing related to affordability,
so, two questions really is:
The differences that you've seen in terms of strategies
among different nations where you've studied [sic].
But also, the issue not only of affordability,
but the distribution. Right, distribution,
how do you get these tests from one place to another?
The real practical aspects of scalability and distributions.
Daniel Dexter: Great questions.
I know that other speakers today will be talking
to these specifics of this amazing programs,
inspiring programs across Africa and the Caribbean now.
So, what I can tell you is that there is
a mixture of lab-based screening.
So, in Uganda they coopted existing
sample transport linguistics for HIV/EID
that U.S. funded for over 15 years.
And they were already transporting dry blood spots
for HIV screening, and Cincinnati sponsored
just taking another one of those blood spots,
and they set up a IEF lab in comparlor [phonetic sp]
and they screened over 100,000 infants
within two or three years;
and they developed some incredible [unintelligible]
data as a result. And they used that data
to develop a targeted screening strategy
to folk in the high burden areas.
Other places, however, they still struggle.
They couldn't scale it to the whole country.
I know that's -- other speakers today will talk about today
what they are doing in places like Nigeria,
using point-of-care testing in the Caribbean.
So, I think there are a lots of things going on,
but I think we are going to move away from strictly lab-based
to a kind of hybrid approach, incorporating point of care.
And the finally questions about distribution,
maybe we need a businessman up here,
but I think there is one place,
one pharmaceutical is the only company in Africa
which makes hydroxycarminide [phonetic sp].
I am not sure, but I think if we could work out ways
to manufacture and coordinate distribution;
it would be incredible powerful to reduce
cost and increase access to test and medication.
[applause]
Swee Lay Thein: Thank you so much for setting the stage
for the next part of the session on screening
for sickle cell disease,
and for this new on screening updates part of our agenda,
we have four panel speakers.
I'm going to introduce them one by one
and invite them to come and sit at the table here.
So, I will introduce all four at one time,
so you will not be interrupted by me when they do their talks.
And the questions and discussion for this part of the panel
will be at the end of the fore talks.
So, first off,
we start with Jennifer Knight-Madden Knight-Madden.
Jennifer Knight-Madden is a professor
of pediatric pulmonology and clinic research
at the Sickle Cell Unit in Jamacia.
She's an examiner and member of specialty
board of the Childhood and Adolescent Health at UWI
as well as honorary consultant
at the University Hospital of the West Indies,
and she will speak to newborn screening in the Caribbean.
Next up, we have Dr. Obi Nnodu, she is a professor of hematology
and blood transfusion and director
for the Center of Excellence for Sickle Cell Disease Research
and Training at the University of Abuja in Nigeria,
and Obi will speak to Africa.
Third speaker is Dr. Raffaella Colombatti.
She is a pediatric hematologist, oncologist
at the Pediatric Hematology Oncology Unit Azienda Ospedale
in the University of Padova, Italy,
and she will speak to well-resourced countries,
mainly Europe.
And our fourth speaker will be Dr. Ana Cristina Silva-Pinto.
She is currently the coordinator
of the Sickle Cell Disease Program
at General Hospital of Ribeiriao Preto medical school
University of Sao Paulo, and she will speak to all low-,
or middle-income countries mainly India and Brazil.
So, let's go and I'll invite the speakers,
one after the other to make their presentations.
Phase three, we don't have the monitor screen
up for some reason please. Thank you.
Jennifer Knight-Madden: Good morning,
if you could bring up my slides, please.
Welcome everybody to Jamacia, I know there are several people
who are here for the very first time,
and we are delighted to have you with us.
So today, I am actually kicking off with the Caribbean,
and we do need the monitor.
Next slide please, oh clicker.
Okay so, the Caribbean is home to about 44 million people,
and we have the highest incidents
outside of West Africa.
But you know, when Daniel talked about context
that's really an issue.
So, we have -- okay, we are going to talk
about different aspects of the Caribbean,
including the geography, the history,
the politics, the economics,
because all of these things actually impact
on what is the best choice in terms
of what you're going to use.
So, I -- [inaudible]
Jennifer Knight-Madden: Can I what?
[inaudible]
Jennifer Knight-Madden: Oh, it's over there.
Okay, well everybody look over there.
[laughter]
Jennifer Knight-Madden: Right, so,
we are going to look at newborn screening
from the point of view of countries that have started,
had pilots, whether they're screening now,
and the methods, and the choices,
and then of course the poll in collaboration.
So, the Caribbean, it sits between
North America, South America,
and Central America in the Caribbean Sea.
So, some places on the internet will tell you
that there are several hundred islands,
and if you go to the Bohemian side,
they say they have several hundred islands;
and then you add in the keys and everything
and you can see that geography is a real problem.
[laughter]
Jennifer Knight-Madden: Because you can't drive,
you have to fly, right. So even in the small islands,
you know, the leeward and windward island,
it's an issue getting samples from place to place.
So that determines what makes the most sense.
Now, just to tell you that the yellow is Spanish.
All right, so the main land around us apart
from the U.S. and Belize, they mostly speak Spanish.
French is spoken in Haiti,
which alone, accounts for a quarter of our population.
And in the overseas territories of France,
and so that's actually quite interesting
because of the politics and economics the best care
is in the French part of the Caribbean.
And the worst care is in the French part
of the Caribbean, because Haiti as I'll show you
is actually the only low-middle income country in the Caribbean.
I find this quite shocking.
But all of those circles are actually high-income countries.
But, you know, when I looked at
what the definition of high-income countries is,
if you add up the income of the country,
divide it by the population, so per capitol,
and it's more than $14.000.00
U.S. dollars, then you're high income.
[talking simultaneously]
Jennifer Knight-Madden: But no,
you compare that to say the U.S.,
which is like $60,000 or $70,000, right?
You can see that,
and to be low income you have to be less than like $1500.
I mean, whoa, yeah so, Haiti is lower middle income,
and the ones that don't have circles like us,
were upper middle income. And this is important,
because it means that we can't get some of the grants,
we can't publish in some of the journals
without paying and all of that kind of stuff.
So, economics does matter.
The other thing is with the languages,
in sickle cell we all talk to each other.
Not very common in the Caribbean,
but in sickle cell it works.
And in transport, I can't see it from here,
because I am old and I have great hair.
[talking simultaneously]
Jennifer Knight-Madden: We're back, oh, thank God.
[laughter]
Jennifer Knight-Madden: So, okay, so $123.00 U.S. dollar
to send a one-pound envelope
between countries in the Caribbean.
So, if did that once a week, you're looking at $6,400
that might not sound like a lot, but that's for one pound.
Suppose it's more than that, right?
The cost goes up and up, so you have to think about that.
Now in terms of collaboration, COSCA
which is the Caribbean Association
of Sickle Cell Associates is a patient group,
and they used to be very active and they had sort of died out,
but they are trying to come back again.
So, we're going to try and encourage that,
because politicians listen to people
more than they listen to scientist.
So, we need our patients to advocate for changes.
The SickKid Caribbean Initiative
that with Cancer as well as hematology
and we've kind of moved on to a different phase,
but they did train two hema[phonetic sp]
pediatric hema people in Jamacia
and a few more around the region.
So, it was, you know, very helpful.
CAREST as I said goes across Spanish, French, English,
so the president is from Guadeloupe,
I am a vice president and our other vice president
is from Cuba, and we've done screening;
we've done pilots in several countries.
Now we have a working group which so far
has the countries involved.
And we can learn a lot from this.
The first thing is that most places have pilot data.
And that's mostly through CAREST,
expect for Belize where we're trying.
But the problem is that people got pilot data when CAREST,
we subsidized, but then they haven't continued it,
because the government hasn't taken it on.
So, in all those countries there are champions
trying to get this done,
but what I want you to also look at is;
if you accept what is on the internet in terms of population,
birth rate and estimated births
as well as the incidents of sickle cell disease.
You can see in some countries
you might have six births a year,
as against Haiti where you have close to 2,000.
So, we need to work on Haiti,
because they are not screening universally.
Right, but in a place where you have 1400 births
that's going to affect which kind of machine you can use.
You don't need HPLC for sure.
So, which one are we going to use?
The choices depend on many factors.
So, is it something you do at the bed side?
Do we have time?
Is it something where you take it from the [unintelligible]
natal unit to the lab in the hospital and do it there?
That may be helpful. Is it quantitative?
Do you have to send it overseas?
So, for most of the small islands,
you would have to send it overseas.
Can you upload the data?
So, from a public health point of view,
you actually have a data base. And what is the cost?
We need to get the cost locally for Hemotype S,C.
That cost is for 20,000 tests, right,
but you can see that were talking about 8 and 10
and up dollars per test, right. And why is that?
It is because we have to bring it to the Caribbean, okay.
So that makes a difference and of course,
how soon you get the results. If you get it right away,
you can get the child into clinic,
whereas, HPLC, you have to go find them afterwards.
So, the collaboration, it depends on the willingness
of the vendors to meet us partway as we heard.
It depends on the willingness of the ministries of help
to make it a priority.
And we want to use Pan American Health Organization mechanism,
where we could buy as a region and then as a rotating fund
which would bring down the cost for everybody.
So, there are many things that we have to take into account,
but we need to get the screening going,
because as we know it completely changes mortality and morbidity.
So, in Jamacia now if you're born, you're screened at birth,
you come to clinic, the mortality is the same
whether they have sickle cell or not.
And that's what we want for all region.
That's what we want for all world. Thank you.
[applause]
Obi Nnodu: Good morning, everyone,
I want to thank the organizers
for giving me the opportunity to make this presentation.
If you can please pull up my slide.
I hope you haven't started the timer,
because I've gone -- [laughs]
-- I've gone through several [unintelligible]
and several seas to come and give a 10-minute talk.
[laughter]
Obi Nnodu: Okay, so, I use this outline,
and so the background is like Daniel said
and also as Jennifer has alluded.
We have a lot of pilot programs; we have had in the past,
which we have not been able to stay up
for the reasons that we have heard, you know, today.
Another thing when, I did the evaluation
or the assessment of the WHO sickle cell strategy
across high budget countries, one thing was very clear.
There was only one country that had budgetary and location
for sickle cell programs
that is the newborn screen in [unintelligible]
that [unintelligible] republic
and think that's probably because of [unintelligible]
means they work.
And so, the budgetary,
they are not making budgetary at location
so when you have [unintelligible]
coming to set up pilot program
and that doesn't go too far once the funding has gone.
In recent years, we've had several initiatives
to address these problem of pilot programs.
One is the American Society of Hematology Consort Program.
The second one is what you have heard now
about the use of point of care technology
that is coming into stream
now across different counties for newborn screening.
And then the third one is the implementation science study
of the Sickle Pan African Research
Consortium which you know is the NIHI
infrastructure grant for sickle cell disease in across Africa,
which I will talk about a little bit later.
But for CONSA, CONSA is a program
of the American Society of Hematology
with African Hematology
to demonstrate the feasibility of newborn screening
and early therapeutic interventions
through African government.
In order to encourage them to do newborn screening
because it's actually possible.
And we have seven African countries,
as you can see on the map.
CONSA is led by national coordinators
with designated clinical catchment areas in each country.
And we have been screening between 10,000
to 16,000 babies a year per country.
And we have the babies are expected
to go into to follow-up care;
but we are finding, as you can see the data later,
that this plan to enroll the babies into follow-up care
has really not been working to well.
Because only 42 percent on average of the babies
are turning up.
And that's for a number of reasons.
So, you screen babies, and they disappear into the bush,
and you're not able to meet up with your goal.
So, we have a trained health care workers
on use of the protocol.
We have trained them on the DBS collections,
ongoing training, retraining on laboratory analysis,
using the [unintelligible] platform,
as well as a quality insurance processes [phonetic sp].
We are also training the health care worker,
because it happens that the CONSA countries
are also the Sickle Pan Africa Research Consortium
have developed a uniform,
multi-level management guidelines.
So, we are training the healthcare workers
on the use of the guidelines.
And also, to know when to refer the babies,
you know, for [unintelligible].
We are also finding out that the mother's do not want
to go to do those big pediatric sickle cell clinics
in [unintelligible] hospitals.
They prefer to stay at the primary healthcare center.
Then we are also training the families on education
and carrying out a community sanitization and awareness.
So, these are some of the things that we are doing,
but when we see the picture.
I will show you pictures of Nigeria.
So, data quality is being taken care of with
on site centralized training to improve documentation,
sample collection,
also to ensure that the samples are properly dried,
especially during the rainy season.
They are stored properly
and they are sent to the central laboratory.
We are also training the models [phonetic sp]
on how to take care of the babies.
We are training journalist on sickle cell disease
so that they can develop programs
through greater awareness.
We are doing community entry, and then we are, you know,
involving the community, going to the chiefs,
and getting them to send the mothers for screening,
and also to make sure that they encourage them
to come for rudimentary to care.
Okay, thank you, so our preliminary works
with the second one is point of care,
and thank God that Daniel has spoken
so I don't have to say too much.
But our preliminary work had demonstrated
high sensitivity and specificity of point of care
even when applied to newborn screening program.
Now you see the number of publications
that have come as a result of that.
So, the third one is the initiative
of the Sickle Pan Africa
Research Consortium newborn screening program
which is an implementation of science-based program.
So, for this one we have six countries,
and then in Nigeria we have 25 sites for this NBS
in Pan African Consortium meet program,
and then within the SFT for CONSA we have 25 sites.
For we are working across multiple sites
with all of these screening programs that I'm talking about.
So within the context of Sickle Pan African Research Consortium,
which you know is an infrastructure grant,
we have a registry of over 34,
000 sickle cell patients in the disease registry,
and what we are using is for this implementation site
we want to use the DBS point of care test,
which we evaluated and found to be feasible
to assess the performance characteristics
of DBS POCT to IEF and HPLC,
and then to use that the DBS POCT
to do a screening within the countries;
and then to look at the context
through a focused group discussion
and in depth interviews.
And then findings are going to be very important
when we design the newborn screening program
for the government
to be able to scale up newborn screening programs.
And then we found out,
okay this is really not the correct slide.
But with regard to patients, parents and caregivers,
they like point of care,
and they prefer post-natal wards and immunization clinics.
And they also want to be educated.
With regards to policymaker,
they understand all that is going on,
and they want us to be able to have more education programs
for the [unintelligible]
and beyond [unintelligible]
that there should be budgetary allocation
for sickle cell disease.
Then, for the part of healthcare workers,
they understand newborn screening should be included,
you know, to lead should be done in order to lead
to better health outcomes and improve,
they understand that. But they are feeling that,
you know, having been spoilt by the HIV screening program,
that the PEPFAR program, that the mothers need incentive
to encourage them to bring the babies,
and they are also advocating for that.
I don't know how that will happen,
but everybody understands that collaboration
between community leaders
and all the different stakeholders
are quite important to be able to get this done.
So, in carrying out, you can see that we've been doing newborn
screening across multiple platforms,
across multiple institutions, across different countries.
So, we find that interaction with the stakeholders
is important
in order to understand the peculiarities of the content,
because we can have an IEF or HPLC-based program,
and you come and put it in a context that it will not work.
Then these barriers that we are identifying
are very important for us to address,
as well as enablers that we have identified
to scale up those enablers in order to be able
to give universal newborn screening.
We also feel that policymakers should --
yeah, okay, I'm going to stop soon.
So, we need more awareness in order
to get everyone to know that this screening exists,
and that's why we are working with the media.
My personal opinion is that after observing the challenges
across our countries in our context,
I feel that IEF is good,
but if you're doing a program that after 23,000 patients,
you're still getting some false positives,
maybe we need to look at some other alternatives.
Also, the observed kit failures in some
POCTs require closer scrutiny. We feel that a DBS point of care
gives us the opportunity for confirmatory
testing by IEF or HPLC with another point,
or with another point of care testing, and molecular testing.
And molecular POCTs could help to resolve,
you know, discordant results, resolve diagnostic challenges,
and would be a natural evolution in order to phenotype
the babies identified with SED for subsequent follow-up
and for future personalized medicine
and for novel treatments.
So, these are my acknowledgments,
and I want to thank you, and this is my team.
Thank you very much.
[applause]
Female Speaker: So, first of all,
thank you for inviting me to give the update
on the European setting, and if you can put my slide on.
It's wonderful to be here for the first time
and meeting many colleagues and friends in person.
While I wait my slide, okay, so what I --
these are my disclosures. What I will present
are some of the European setting specificities,
guidelines that we have currently in European countries,
and the current situation of screening,
some of the challenges of how to prove
that NBS has an impact on outcomes and benefits,
and also the challenges of the communication of the results
to affected children and carriers.
So, when we talk about Europe,
it's a geographical but also political area,
and every country has its own health system or systems.
But there are common grounds for protocol, politics,
and funding, especially within the European Union,
which, as you know, has U.K. outside of it since a few years.
We have a population of around 450 million people,
and 38 of these were born outside Europe.
And this, of course,
has an impact on screening and diagnosis,
also considering that these people mainly come from areas
where mutation of the beta and the alpha globin gene are common
and are of a huge variety.
And this increases the challenges for,
especially in the future,
for different genotypes and phenotypes that can appear.
Now, the main four countries are Germany,
France, Italy, and Spain,
and you can see where the immigrants come from.
And as I said, they come from the Middle East,
Sub-Saharan Africa, and Southern Asia.
Number of newborns in Europe, 3.8 million.
So, this is our catchment population,
although the fertility rate is different,
it's highest currently in France,
which has a population of around 30,000-36,000
sickle cell patients estimated. So, what about the guidelines?
The U.K. was a pioneer in developing guidelines
for newborn screening, but every country
now has national guidelines in their own national language.
And we published a consensus in 2018
recommending universal newborn screening in all Western Europe.
Recently, the British Society of Hematology
included a guideline not only on a newborn screening,
but prenatal and antenatal screening,
so a comprehensive guideline.
And funded by the European Commission
and the European Reference Network
for Rare Blood Disorders,
the national guidelines, eight of them,
have been translated in English.
And now there's a big group in Europe
of specialist pediatric hematologists
working on the harmonization of all diagnostic pathways,
but also clinical pathways for acute and chronic complication.
And we hope to finish this work next year,
and so share with everybody.
What about the current situation?
Well, the U.K. has national antenatal and newborn screening.
There are several national universal
newborn screening in Spain, Germany,
the Netherlands, Belgium, and France overseas territories;
national targeted in France; national universal prenatal,
antenatal, and preconceptual in Greece and Cyprus;
antenatal targeted in Denmark.
There were some pilots conducted in Italy,
Portugal, and Ireland,
which unfortunately have not yet been able
to implement national newborn screening,
even though you can see Portugal has the highest frequency
of sickle cell disease estimated in the newborn population.
We must not forget that in Europe,
sickle cell disease is considered a rare disease.
But this means that patients and individuals
with sickle cell disease risk of being neglected more than once,
because they are rare,
because they are immigrant or descendants of immigrants,
and because they have a different color of the skin
compared to what is considered the average European.
This is just a situation of Italy,
sadly, because of lack of consent
among the European hematology community,
in spite of the figures that you see
that are higher than other countries,
we were not able yet to get newborn screening
at a national level, only regional level.
What about the methods?
Some countries use cord blood; others use heel prick samples.
The method used -- almost all countries use two tests,
but Spain uses only one test.
And the confirmation is performed only when the patient
is referred to the pediatric hematology oncology unit.
And there have been some nice pilots
with mass spectrometry and genetic analysis,
for example, mass spectrometry in France or genetic analysis
and mass spectrometry in Germany.
I found very interesting the one that's unifying the screening
of immune deficiencies, muscular atrophy,
and sickle cell disease together,
because these diseases were recently
added in the newborn screening program
in several countries, Italy included.
What about the data registration and numbers?
There are several evaluation programs on the coverage.
The best is the U.K., again.
But what is really a driving point in European countries
is prospective data collection.
Patients are screened, but then they are enrolled
in comprehensive follow-up programs,
and now they're enrolled in national registries,
or the countries that don't have them in the European registries.
And there has been harmonization
of the definition of the phenotype
and the complication in all European countries,
and we hope this will give information in the future.
We do not have European numbers
on the overall number of newborns screened yet,
only national data.
And the estimate is more than 60,000 individuals
living in Europe.
And these are just some of the examples
of the output in data
of the hemoglobinopathies screening programs
and registries from a few countries.
Now, I am going to highlight Germany,
because Germans always, when they start something,
they move forward. They started screening in 2021,
already have 11 laboratories that are active,
screened more than 1 million babies,
and everybody's enrolled in a registry.
And what they manage with this wide collection of data
and having in a few years insight in the disease
natural history in their population --
so the genetic characteristic,
the phenotype of certain manifestations.
So, in effect, coupling screening
with data collection is important.
Is communication of the result to affected families of children
affected is mandatory,
but of the carrier status is not allowed in Germany
due to the history during Second World War.
And there have been evaluation
of recent communication strategies,
both to the affected children, families, and the carriers.
So, it's important to continue this evaluation.
What about the outcomes
and the benefits of newborn screening?
Are we really capable of proving that it's beneficial?
Now, I was surprised by this sentence
that was presented at the National German Hematology
Screening Meeting last year:
Screening reduces early morbidity and mortality,
but to what extent is unclear
and will probably remain unclear.
Is it true, or have a way to demonstrate this?
Now, in France, for example,
they demonstrated that there are many failures
in the targeted screening programs.
So, this is a way to address the problems.
But another way is looking at the global disease burden
that was cited before.
We were surprised that in the U.S., sickle cell disease
is not one of the most common causes of death under five.
But look at Europe. In many European countries,
sickle cell disease is between the third
and the sixth cause of death,
even in my region, which is the northeast of Italy.
So, is it because this data was collected
before many newborn screenings scaled up,
or is it before we have other issues?
I think we will need to reassess this in a few years.
Nevertheless, how did countries in Europe
look at the benefits of newborn screening?
This is a paper that came out recently
in American Journal of Hematology by the Dutch.
They have a newborn screening.
They have a national prospective data collection
that allows them to evaluate how good their care is.
74 percent are treated with hydroxyurea.
And in fact, there was a reduction
in mortality from before to after.
But morbidity remains high,
especially in the 0-4 age group with acute events,
except acute chest, with worsened over age,
and also the chronic complication.
Similar figures in Spain.
Newborn screening, prospective data collection,
and comparison of mortality and acute morbidity
with other newborn cohorts.
Reduction of mortality, but same morbidity. France.
France does not have a national universal newborn screening,
nor a national registry.
So, this was a retrospective review.
Nevertheless, the survival was high.
HU was prescribed in only 30.7 percent.
But the conclusion was there was still a high risk of morbidity
and burden of disease.
So, my conclusions are the NBS programs are developing
and scaling up in Europe,
although with different strategies.
But this might not be bad,
because there is no one size fits all,
even for neighboring countries.
Data collection at national and European level is prioritized
and is being harmonized to allow comparison of data.
There is the need to improve communication
and patients' communities' involvement.
And, of course, we still have to overcome some challenges
of cost,
prejudice, lack of uniform voice among stakeholders,
and different other priorities in times of budget constraints.
So, I thank the European colleagues working on this.
I probably forgot somebody.
We have a long road ahead, which might not be straight,
but nevertheless, we need to continue walking.
Thank you all for your attention,
and of course, our patients and patient association.
[applause]
Female Speaker: I'd like to thank the organizers
for inviting me to talk about newborn screening in Brazil,
my country.
And I would talk a bit about India in afterwards.
Well, in Brazil, the National Screening Program
was launched in 2001.
And in 2005, the National Program for Sickle Cell Disease
and Other Hemoglobinopathies was launched.
And in 2006, our scientific committee was funded.
And this scientific committee wrote
our National Protocol for Hydroxyurea
Use, Transfusion and Iron Chelation,
our TCD screening program, and bone marrow transplantation.
So, we have all these programs since the last decade.
Well, I don't know if you all know
that Brazil has a public health system.
It's called SUS, Sistema Único de Saúde.
And its assistance begins in primary care,
actually in neonatal screening,
responsible for first procedures of prevention,
such as prophylaxis with penicillin and vaccination.
And it goes all the way to reference centers
that will take part with TCD screening,
magnetic resonances, and bone marrow transplantation.
Data from newborn screening started to come
and to be published in 2007. And with this map here
that shows the prevalence of sickle cell trait,
of course, its prevalence is bigger in Bahia,
the state of Bahia, the first capital of Brazil
when it was part of the Portuguese empire.
The prevalence ranges from two percent
in the very south of Brazil,
where we don't have that much influence of our descendants,
until six percent.
In my region, it's 2.5 percent, the state of São Paulo.
And data -- this is new data already, data from the CISNET,
the National Registry for Newborn Screening,
ranges between 1,500 to 2,000 newborns
with sickle cell disease every year in Brazil.
But this may be underestimated,
because, as you will see in my presentation,
the coverage of newborn screening,
which is universal in Brazil, is not 100 percent.
So, talking about newborn screening,
it started in 2001 in the southeast region.
That means state of Minas Gerais,
Rio de Janeiro, and São Paulo State,
the three most populated and richest states.
São Paulo has three screening centers,
because it has 45 million people living only in São Paulo State.
Brazil has 200 million people.
One-fourth, almost, lives in São Paulo.
So, it's a huge state when it comes to numbers.
It's all 100 percent performed by HPLC nationwide.
And we finished training and implementing HPLC
for newborn screening in Brazil in 2013,
with at least 80 percent of the newborns coverage.
We divided the implementation of newborn screening in Brazil
into four phases. Now we are heading to the fifth.
But first phase phenylketonuria congenital hypothyroidism.
Phase two, sickle cell disease. Phase three, cystic fibrosis.
And phase four, congenital adrenal hyperplasia
and biotinidase deficiency.
Now, as you can see, and you can follow the Brazilian map,
this is the evolution of neonatal screening program
in Brazil.
As you can see, from 2010 until 2013-2012,
there were some states in phase one with light blue.
But in August 2013, they were all screening untill phase two,
at least which covers sickle cell disease.
And in 2014, all the nation was screening until phase four.
And the coverage ranges.
In São Paulo State, it's above 95 percent.
So, in São Paulo State,
almost 100 percent of the newborns are screened.
But it's not the reality of our entire nation,
because Brazil is so big.
We are considered a middle-income country.
But we have, inside Brazil, low-income regions,
middle-income regions, and high-income regions.
So, it's very complex to really have an orchestra
of all the things we have to do in sickle cell
with the differences among states.
But, as you can compare, the first map I showed you,
there was no data regarding the center of Brazil in the north.
And now this presentation includes this publication
of 2018,
that we already have some data from the center-west
and the northeast part of Brazil.
Of course, the state of Bahia is the most prevalent one
because we have a lot of influence
of Afro-descended people there.
But we are facing some challenges.
Newborn screening faced a drawback in Brazil
since 2019 with the switch of the government.
So, we had a government between 2019 and 2022
that didn't take any care of sickle cell disease,
didn't look at minorities,
and didn't give almost any budget
to our public health system.
With reduced budget, the sickle cell screening program
was reduced to less than 80 percent coverage
in most of the states of
And our national registry the poorest regions,
north and northeast of Brazil.
And our national registry is called Web Hemoglobinopatia,
it's still incomplete. But since last year,
we're putting many more reference centers on board.
But we are concerned about the disparities
that we face in our country,
with poor health support for sickle cell patients
in the northern states of the country.
Well, but there is light at the end of the tunnel.
We have a new government since last year,
and that raised sickle cell disease
as one of its priorities,
re-established our scientific committee --which I'm a member.
I was a member, and then they shut down the committees,
the scientific committees. We are all volunteers.
We don't cost a dime for the government,
but they shut down in 2019.
So, everything was in darkness, and now it restarted again.
And we started training neurologists and doctors
to perform newborn TCD
because the federal government bought the machines,
but the doctors didn't know how to do that.
So, we started in our institution,
with a collaboration of the Department of Neurology.
We had many, many doctors coming from all over the country
to be trained to perform a transcranial Doppler.
And last year, we finally had our National Sickle Cell
Disease Congress, sponsored by the Ministry of Health.
It was supposed to be every two years,
but the last one was in 2017. And then darkness came,
and then now we are out of darkness again, thank God.
And next year, we're going to have
our 10th National Congress in September
in the center of Brazil,
to try to develop sickle cell disease
in the center-west part of the country.
Thank you very much for your attention.
[applause]
Well, yes, I changed my presentation,
but they didn't put the new presentation in,
because I just realized that I was going to talk
about India yesterday night.
But, well, I don't know if you can have the presentation
I uploaded this morning.
Male Speaker: You uploaded it down there?
Female Speaker: No, with that lady there,
that she's going.
Yeah. Well, yes. See, I updated even with the flag.
[laughter]
Okay, let's go.
Of course, I know more about my country,
but I was surprised to note that it is estimated
that 15 percent of the world's neonates
with sickle cell disease are born in India.
Two-thirds are born in Africa, 15 percent in India,
so it's almost, what, 80 percent Africa and India together.
So, it's a very important country to look for.
And there is no national neonatal screening program
for sickle cell disease yet.
But it started, as this publication
was published in 2018.
This presentation shows a few newborn screening programs
that has been initiated in the last five or six years,
so in the last decade.
As you can see in this map of the Indian country,
you can see some regions
where they implemented universal screening
and targeted screening,
mostly in the northern part of the country.
And I was surprised to see the prevalence.
Those are the states and districts
at the beginning of the table. But then number of screening --
I'm sorry, well, number of screened babies
and the percentage of the S mutation -- I was surprised.
One region was 45 percent, the Maharashtra Nagpur district.
Look at that, 45 percent of sickle cell trait.
That's a lot. And compared to Brazil, I was shocked.
And number of sickle cell disease patients
in this last table. So the numbers are pretty great.
And, of course, this is a pilot in some regions.
But since 2010, it has been initiated.
They already screened over 18,000 babies
and found almost 3,000 sickle cell disease individuals
and 300 babies with sickle cell disease
and 3,000 sickle cell trait.
But now, last year, they launched that program
that the first speaker told us --
the Sickle Cell Anemia Elimination Mission.
And their aim is to turn neonatal screening universal
in all Indian country.
And so, I think they have started with,
and then they are trying, as you said,
many kinds of newborn points of care to see which one is good
and not so expensive to become nationwide use.
So, I think we are going to hear a lot more
about India's newborn screening the next few years.
Thank you.
[applause]
Female Speaker: Thank you so much to the four speakers.
It's striking to note the similarities,
but especially how different the programs must be.
And we must realize that the different methods used
and continue to learn from each other.
It really was four great talks. So, thank you so very much.
And 10 minutes is not a long time to speak for regions.
I know Jennifer could speak maybe an hour on Jamaica alone
because of the wealth of information
and what we have learned over the last few years
about newborn screening
and what strategies work and what don't.
Open for questions.
I'm trying to follow in the Zoom.
I'm not seeing much questions.
I'm only seeing requests for CMEs.
Yes. Go ahead, Dr. Little. Is there a mic there?
Jane Little: Thanks. I'm Jane Little.
I'm from the University of North Carolina.
That was really so enlightening.
I had
[inaudible]
-- if you could comment about that, that'd be great.
And the second question I had is --
it's so annoying to me that we have newborn screening
and then we don't have registries.
And I've been boring people about this, I think,
for a very long time.
And I wonder if you guys could talk about ways
that we could try to link newborn screening
to registries that we can all learn from
because we're not learning from each other.
And I think that's just a shame.
And you'll hear me say that again. Thank you.
Female Speaker: Can you -- I don't want to mess with this.
Can you hear me?
Do we have problems with the microphone?
You want to come here? No. No. Are you listening?
Yeah. We're listening. Okay.
Well, of course, politics is --
they are in charge of the money, of the budget.
So, when it comes to newborn screening,
which we were talking about, it costs a lot of money.
Especially if you're talking about in the whole region
or in a very big country, universal screening,
it takes money, and it takes expertise,
and it's a continuum. That's the problem.
You can launch the program, you can put some budget,
but it needs to be lifelong.
And the problem is if you switch government
and the government has the power to just stop
giving money to the program, the program is going to die.
So, whether you put this aside
of what the government can do -- no.
By law, you need to give 10 percent
of the GDP to healthcare.
But otherwise, you are going to be in their hands.
And if it happens like we had in Brazil,
a government that doesn't care, actually,
to the health of the people.
Actually, he told that there were too many
Brazilians reading Brazil. It's a very big problem.
The programs just were paused or stopped,
especially in poor states.
The state of São Paulo didn't change
because it's the richest state we have.
But in some regions where the federal government budget
is essential, it will not.
So, vote cautiously. Voting is important.
Public health systems is important
because the people cannot afford treatment.
We perform bone marrow transplantations
for free in Brazil. Not for everyone,
because we don't have that many reference centers
for bone marrow transplantations.
It's our goal to start new reference centers
for bone marrow transplantations because gene therapy is coming.
Yes, but gene therapy --
you cannot perform that in an outpatient.
So, you need to have hospital beds and reference centers.
So, we are trying to start with reference centers
for bone marrow transplantations
to have at least one reference center in each state.
Because what happens today,
everyone comes to São Paulo State to perform BMT.
And they have to wait in a long line
because we have the state of São Paulo people
and the other states coming together.
So, if we have one reference center
like we do in newborn screening,
one reference center for newborn screening in each state,
one reference center for sickle cell disease care,
and one for bone marrow transplantation,
I think we're going to be good to go.
Female Speaker: I think we'll stop there.
I know you had another bit to your talk,
but we are 20 minutes delayed, and we have a long program.
So, I'm going to encourage -- I'm sorry to cut short --
because this has really been an exciting panel and these talks.
We need to speak about the registry, et cetera.
But let's mingle.
We have a 10 minute only for a break.
We are hoping to cut off by seven to eight minutes
so we can try to get back on schedule.
So, thank you so much,
all of you, excellent presenters,
for leading the morning, as well as
[inaudible]. Thank you so much.
Let's have a short break -- 10 minutes.
Zoomers, as well, can take a stretch break,
and we'll meet back at 10:30.
Female Speaker: Okay, welcome back to session two of day one.
We all know how complex sickle cell disease is,
affecting multiple organs,
and collaboration across disciplines is so important.
And as I come to understand sickle cell disease,
it's really very much a biomechanical disease,
even though we keep talking about the root
cause being hemoglobin S polymerization.
But the rascal that carries out the damage
is really the red cells and what it does,
the red cell membrane skeleton.
So, I'm so delighted to actually chair this session
because we want to talk about
how we can clinically translate bioengineering and biophysics
to better understand
the pathophysiology of sickle cell disease.
And I also have a slight change in the program.
Dr. Alan Docter has to give another virtual presentation,
so he will follow Wilbur.
But first, let me start with Dr. Wilbur Lam,
who is a tenured professor of pediatrics
and biomedical engineering at Emory University
and the Georgia Institute of Technology.
Wilbur is also the associate dean of innovation
at Emory University School of Medicine.
Wilbur is no stranger to SCIF, and I'm so delighted
that he could make it in person this time.
Thank you very much, Wilbur.
The other thing I want to remind all speakers is,
we have to be very strict with timing. Nona [phonetic sp]
will show me when time's up after 25 minutes.
I will actually physically yank you off,
so be warned,
because we cannot eat into other speakers' time.
Thank you, Wilbur.
Wilbur Lam: Okay, I better hurry.
I don't want to be wrestled by Svila [phonetic sp].
So thank you so much for having me,
and thank you for letting me talk about a topic
that I personally think about quite a bit,
and also to show that there's a growing community in this field.
So, I'll talk about this technology called microfluidics.
And it's something, like I said,
I've certainly been thinking about for a while.
And I should give my disclosures,
first of all, that I am a co-founder
of a couple of diagnostic companies.
And also, that this talk is actually not done just by me,
but a couple of my close collaborators in Atlanta --
Dr. Vivian Sheehan and also Kendall Williams
and Bev Rogers, who are pathologists.
So, clinical laboratorians
who work in the field of diagnostics.
And that's actually one point that I want
to make-that we here, as clinicians,
need to work with our pathology colleagues
when we're really talking about new diagnostic tests,
because this is what they think about 24/7,
how to bring something to practice and implement it.
Okay. So, you know, Sui-Lei [phonetic sp],
when you invited me to give this talk,
it really reminded me of when I first gave it.
So this goes back to 2013,
when you invited me to give a very similar talk in London.
And this is June 7, 2013. And I believe when we met,
Sui-Lei, you looked at me and you said,
"You look like a schoolboy."
[laughter]
I do not have that problem anymore, right?
So, in fact, just the other day, one of my grad students and I --
my brand-new grad student and I -- we were walking,
and we have these doors at Georgia Tech
where you put your key card. And I put my wallet on.
My wallet dropped, and she sees my driver's license.
She sees my date of birth.
She turns ashen. And I said, "What's wrong?"
She goes, "I didn't know you were that old."
And I can tell she's already thinking,
"Oh my God, I need a new Ph.D.
advisor because this guy's going to be dead by next year."
Anyway, so thank you for the opportunity.
But it also shows that this is stuff
that we've been thinking about as a field for a while.
We talked about a technology called microfluidics
and how that could help us move the field of sickle cell disease
and potentially as a biomarker or diagnostic tool.
So what is this? What is this weird word?
So, really, all it is is just a small gadget
in which it deals with small amounts of fluid
in these little tiny channels. It's about plumbing.
And when you shrink down, it allows a few things.
It reduces reagent consumption,
increases speed, analytical performance,
allows automation, integration of different complex workflows,
maybe multi-parameter testing,
and the possibility of point-of-care clinical testing.
So, there have been technologies that have been moved
into clinical implementation, and large companies like Abbott
have already made products in this space.
From the research side, we're already all doing this.
So you might have heard of spatial transcriptomics,
single-cell sequencing.
Those all leverage these technologies
that are technically called microfluidics.
So what are microfluidics?
Well, really, it's the technology
that you all have right now on your laptops,
on your cell phones. It's microchip technologies.
Microchip technologies that have been pivoted
and changed a little bit -- modified a little bit.
Instead of metallic silicon chips,
we're now dealing, in general, with a polymer.
With typically, the most common polymer
is something called polymethyldialoxane, PDMS.
And it's transparent.
It's really good for things like microscopy.
It also happens to be gas-permeable,
which is nice for us in sickle cell disease
because then we can mess with oxygen tension.
It can be functionalized.
It can be integrated with other sensors.
One not-so-great thing, which I'll come back to later,
is that this is not so great for manufacturing.
Now, again, if we're thinking of an actual product,
those are important things to consider.
Okay. So this was my very first foray into using microfluidics.
This is something I made as a grad student many,
many years ago now.
And I took blood from a healthy subject
when I was in grad school.
No one wanted to sit next to me in grad school
because I always stuck them for blood.
And then I took the same device and brought it to my clinic
when I was a fellow in pediatric hematology/oncology
at University of California, San Francisco,
and I just perfused a sickle cell patient's blood --
whole blood -- and you can see right then
and there, wow, there's something here.
That these relatively simple devices,
when put under a microscope,
can recapitulate aspects of sickle cell pathophysiology.
Okay, so can we translate these technologies
to study sickle cell disease? And now the question is,
can we even bring them to the clinical side?
Can we make clinical decisions with assays like these?
That's the big question that the field is trying to deal with.
So, a little bit of what my lab does,
since I've been in Atlanta the last 12 or 13 years now,
we study the biophysics of hematology.
And we also create new technologies
and new devices with microfluidics, and so forth.
We're always very interested
in trying to translate these devices
that we make into point-of-care diagnostics,
even home-based diagnostics,
and then bringing them back to the patients
that I see on a regular basis.
So, that's the space that my laboratory lives in.
And this was something I originally talked about,
Svila, in 2013. It was a technology we developed
in which we grow endothelial cells
in these very, very small channels.
The lab -- not myself anymore, but the guys in the lab --
can almost grow endothelial cells in anything.
That was pretty crazy.
But this was a study that we published
in 2012 in collaboration with Russell Ware.
And what we showed is that, yeah, in general,
these things kind of work.
They're good to study aspects of sickle cell disease.
What you have here is blood from a patient
with hemoglobin SS on the left, blood from a patient
also with hemoglobin S on hydroxyurea.
We now use these videos in our clinic in Atlanta
to show patients, like, "Look, this is what happens.
This is why hydroxyurea is good."
Okay, great.
So this microvascular chip platform
can serve as an in vitro model of disease.
That's great for different applications.
So, fast forward a little bit. What else can microfluidics do?
Well, it's really good for research.
In fact, there's a brand-new paper
that's still in press in Blood. I won't belabor the point,
but we have actually used microfluidics
to figure out a new aspect of pathophysiology
in sickle cell disease when it comes to vasculopathy.
I can talk to you about this for an hour.
But the bottom line is that, using microfluidic platforms,
we have shown that the biophysical alterations
of sickle cells themselves
enable them to marginate more toward the vascular wall,
therefore causing the endothelial cells
to become more inflamed.
So, this is a new aspect of vasculopathy
that is distinct from vaso-occlusion,
that's distinct from hemolysis, it's distinct from adhesion,
but maybe it allows us to think about how this vasculopathy
is connected with sickle cell disease.
And maybe it can help us explain the pathophysiology
of the underlying vasculopathy that then predisposes to stroke,
like in our kids.
So, again, great platform to answer
some of these research questions.
And this was done by Christina Caruso,
who's a junior faculty in the lab
and a pediatric hematologist/oncologist
who will be moving to New York.
Sad for us, but she's starting her own faculty position.
Okay. So, what value, then,
do these assays bring to our field in hematology?
Again, I told you, great as enabling research.
They're good in vitro disease models.
They're also very good at using --
if you have questions about specific mechanisms of action.
You can now take a reductionist approach
to really tease out some of these biophysical aspects
when there is a drug that's related.
So, pharma has come to us recently --
all the pharmaceutical companies that you guys know about --
when they're wanting to explore questions
about their therapeutic agent in sickle cell disease.
We've had several contracts with them.
Great as pre-diagnostic tools.
And I'll focus on that prefix, "pre."
And in informing clinical trials.
So, Marcus Carden, when he was a fellow in my laboratory,
did some really interesting experiments using microfluidics,
showing that when you give different IV fluids --
different clinically available IV fluids --
the adhesion and the deformability actually change.
And that actually begot several large clinical trials.
And the most recent one was just published,
I think, this month, really showing
that, yeah, normal saline
compared to lactated Ringer's is actually bad.
So, microfluidics were a great way to generate a hypothesis.
So, all this is great,
but the question really is clinical translation, right?
Can these things really be used to inform
clinical decision-making?
So the field has gotten much bigger.
So I have plenty of friends now in this field,
many of whom you collaborate with them.
If you look at the number of papers
published in sickle cell disease and microfluidics,
it's certainly come up since I've been in this space.
So, there's groups like Nicola Karr [phonetic sp]
and Conrad in Brazil, Umut Gurkan's group in Case,
and Sarah Du in Florida, Dave Wood in Minnesota.
All of these are people in our community
that are all developing
and leveraging microfluidic technologies
to really push the field for sickle cell disease,
but also constantly asking the question,
can we use these things for clinical decision-making?
Luckily, there's a few groups
that have already started companies to work
with folks like yourselves in the industry.
Patrick Hines has Functional Fluidics, Umut's Biochip Labs --
all are trying to leverage these devices
as assays that can try to inform clinical decision-making.
All right. Now, I'm going to give a slight detour
about things that I've done that I think is hopefully related.
So I lead an NIH-funded
via the National Institute of Biomedical Imaging
and Bioengineering,
where the good Dr. Platt now works.
And we are part of the NIH's Point of Care Technologies
Research Network. We are here in Atlanta.
We have a very, very, very long name.
Our nickname is just ACME for short, so we just say ACME.
So, the reason I mention this is because before 2020,
we were charged with helping other folks
develop point-of-care tests for numerous applications,
not just sickle cell disease. And then this happened, right?
So I was called to a Zoom on April 20, 2020.
Okay, so it was a while ago now.
And remember, Zoom had just started.
So I remember waking up.
It was like 07:30 a.m. I was straight out of bed.
I go to my e-mail. It says, oh, NIH Zoom.
And I push on it. Remember, I just got out of bed.
I don't have a shirt on, right?
So on the other end was this guy.
So, former NIH director Francis Collins.
And I'm like immediately, oh, my God.
I couldn't find the button to turn off the camera
because it was brand new.
I'm like ducking under and I'm screaming to my wife,
get me a shirt.
So, what had happened was Dr. Collins explained to me
that there's this new disease. We need new diagnostics.
We need point-of-care diagnostics.
And you are the director of your center that's part
of our NIH point-of-care technologies network.
We're going to create a brand-new initiative.
And it was going to be called the RADx Initiative --
Rapid Acceleration of Diagnostics.
So, what that meant was that at the beginning of the pandemic,
the United States government actually turned itself
into almost a venture capitalist
when it came to identifying, vetting,
and then mass-producing rapid diagnostic tests.
So, if you think about how long it takes,
and this will be relevant at the end of my discussion,
how long it takes a diagnostic test
to actually come to market, once the biomarker is validated,
it takes, on average, 10 to 15 years to get to market.
That is the average.
What RADx's goal was to shrink that down to one to two years.
And frankly, it actually worked.
But what it entailed was this kind
of very venture capitalist-like perspective
where they took in all applications.
We had over 1,000 applications,
and there was going to be this vetting process.
Phase 1 is going to be a Shark Tank-like process,
and that was named by a couple senators
who, I guess, watch TV a lot,
and they said, let's do it like Shark Tank.
And then there's validation.
So, that's where our center came in,
because we were part of the network,
and our core capabilities
was about technology and engineering.
So, we functioned as the verification center
for the entire NIH when it came to COVID-19 diagnostics.
So, every single COVID rapid test
that entered the U.S. market came to us first for evaluation.
So how did we do this?
We created a huge infrastructure, a giant biobank.
We actually have the largest COVID-19 biobank
in the country at Emory and at Georgia Tech.
Every single variant, every single genetic mutation.
We were one of the first to use our biosafety level 3 labs,
and we were also one of the first
to actually go out to the communities
and collect all these samples, all drive-thrus and so forth.
And importantly, and this goes back to my conversation
I'll talk about a little bit,
we learned about how to assess technology readiness level.
Like, when is something mature enough to go to the next step?
And usability and human factors we found is so important,
and I'll come back to all that.
Okay, so this is just the summary
of everything that we've done for RADx.
The point is, we remain very active for other diseases.
Right now, we're helping them deal with H5N1,
you know, bird flu.
That's a major issue, but also related to hepatitis B,
which is now Francis Collins' big --
hepatitis C, excuse me --
one of his big initiatives with the Biden administration.
And what we've learned, however, is some really important things
that can translate to other diagnostics,
including those with microfluidics.
So, RADx did what it was supposed to do --
force fast failure.
But out of the ones that failed, why did they fail, right?
What lessons we have learned here?
So, we did an analysis here.
I'm going to speed up a little bit,
but the bottom line is that we found
that out of all the tests that failed,
and these are COVID tests, the main reason they failed
wasn't that they didn't work technologically;
it was that they couldn't be manufactured easily.
The design wasn't so great that it allowed the user
to have it be messed up,
you know, mess it up all the time,
and usability -- those were really the key factors
that would kill the technology.
It wasn't how sensitive it was, it wasn't how specific it was --
it was usually these things, right?
So, we're really taking those lessons learned.
So we're also learning quite a bit about human factors.
So, I think, we had a few speakers talk
about lateral flow tests, and so forth.
Well, you can actually mess that up
in terms of its manufacture
if you mislabel the control and test line.
The bottom line here is that we have started to create
a whole set of knowledge when it comes to human factors
and how it can be applied to point-of-care tests,
including those for microfluidics.
So, now we're collaborating with the FDA,
who want us to help them craft microfluidic guidelines
in terms of bringing those technologies
to clinical practice,
and especially at the point-of-care.
So, sometimes simplicity
actually trumps technological features.
That's something that we've learned quite a bit,
and we're RADx.
Okay, so how do we bring that all back?
You know, what are we thinking of when we talk about,
like Sui-Lei said, how in hematology,
and especially in sickle cell disease,
we're dealing with a mechanical disease, right?
So, there's some issues here.
And we were part of a workshop in 2021
that the NHLBI wanted us to really talk about.
You know, how do we bring microfluidics
to the forefront of clinical medicine?
And a group of us, led by myself,
Dave Wood, and Umut Gurkan, we actually convened
all the brain trust of microfluidics engineers
and hematologists,
and we came up with a framework about,
well, what are all the issues?
So, I mentioned some of them already,
but what you can see in general here,
in the interest of time,
is when you're dealing with blood
and you've got to load blood into something,
you've got all these pumps and you've got these pipes --
that can screw things up.
When you're dealing with all these complex interconnections,
that can screw things up.
When you're designing things that aren't for the end user
in mind, for example, if it's at the point of care,
then it has to be designed for someone who operates there,
maybe a nurse, maybe even a clinical technician --
not someone with a Ph.D.
So, those have always been problems
that have stymied the field. We've identified these problems.
Often, when we're talking about microfluidics, we show it off.
We show, "Oh, look how small it is.
Look how great it is." You know what?
There's usually a lot more other equipment around it.
In fact, one of my friends at Penn, Scott Diamond,
he says the joke about this field
is that we call it "lab on a chip,"
but it's actually "lab around a chip."
So, sometimes there's a giant microscope.
Sometimes there's like a $100,000
to $500,000 microscope that's attached to it.
I'm like, "Well, that's not quite point of care."
So, those are things that we're talking about.
Also, now that we're dealing with blood --
and if we're talking about things like viscosity,
which we talk about all the time in sickle cell disease --
guess what?
That's actually really complex,
especially when you're talking about the microscale,
which, inconveniently, is the point,
the site of pathophysiology or a lot of the pathophysiology
we think is going on with sickle cell disease.
So, here's some old-school fluid mechanics for you.
So, all I need to show you is that blood viscosity changes
when you're at different parts of the circulation.
The faster the blood flow is, the lower the viscosity.
The slower the blood flow is, the higher the viscosity
because these red cells clump together --
the rouleaux formation.
You guys might know about that. That actually affects viscosity.
And also hematocrit, as you can imagine.
When hematocrit changes, that is the major driver of viscosity.
So, when you suddenly are dealing with patients
that have different hematocrit or different hemoglobin levels,
guess what? By virtue of that, their viscosity changes.
And, also, by the way, the diameter of your channel --
the diameter of your blood vessel --
that affects your blood viscosity.
So, when you're at the macroscale,
the word viscosity doesn't even mean anything.
You don't even know what it means.
So, that's one ding.
Another ding is that sickle cell disease, in and of itself,
by virtue of having hemoglobin S,
further increases complexity of blood viscosity.
So, I'm going to talk about that.
So all this means is that clinical implementation
of sickle cell microfluidics is hard.
So, when we brought in our clinical laboratorians,
they said, "Okay, well, here are your guidelines here.
You really need to think about all these things."
And I'm going to have to just gloss over it
in the interest of time.
But you need to think about not only
when you start your proof-of-concept pilot studies,
which many papers have done and many groups have done.
We always start with a great paper, like,
"Look what we could do." But now you need to optimize.
Now you need to characterize things like sample stability.
You now need to think about precision.
You need to think about analytical sensitivity
and specificity. What's your reference range?
What's your limit of detectability?
Where's your linearity range?
So these are all things that clinical laboratorians
think about all the time.
So, if we think about the trouble spots
with sickle cell disease --
well, first of all, we don't even know
what a good negative control or a positive control is.
We can think of some,
but implementing them is not so easy.
Your COVID test has a control line,
has a positive control line.
We need to do something similar to that.
We haven't gone to that. One key point is variability.
Variability is actually a hallmark of the disease,
whether you're talking about interpatient variability,
intrapatient variability.
You draw a blood sample from a patient on Monday.
You draw another one on Thursday.
It could be completely different.
And we're even learning that there's intrasample variability.
You look within one milliliter of a blood sample
compared to another milliliter of the same blood sample --
it could be completely different.
So, these are things that we're challenged with.
But having said that,
the field is moving toward that direction --
doing a lot of what I call boring but important studies:
user repeatability, looking at multi-site comparisons,
the CV, coefficient of variability.
These are all things that the field
is starting to move towards --
looking at sample stability and storage.
And yeah, we can demonstrate, for example,
with an adhesion assay based on red cell adhesion to laminin,
stuff that many of us have known,
many of our own researchers in this audience
have demonstrated decades ago.
Translating it to a microfluidic assay --
we need to do that. So, we're able to show
the genotypic differences that we expect.
And then we're even moving toward devices,
like in this case from Ümit Gurkan's lab,
where we're able to maybe obviate the need for microscopy,
starting to integrate sensors into this.
And again, getting a sense of whether or not
we can differentiate different genotypes,
and hopefully even getting a sense of,
could we start to predict who has VOC versus not?
From our own laboratory --
I'm wrapping up the last couple of slides --
we also have been working with pharma
to look at efficacy of new therapeutics.
So, in this system here,
we have our endothelialized microfluidic,
and we're staining white cells and blood cells.
And in this case, it's a new P-selectin inhibitor,
a synthetic PSG01 glycopeptide.
And we're trying to do these studies
where we can differentiate a signal
from these different therapeutics.
Okay. But again, controls and standards are needed.
Okay. So, one solution for microfluidics is --
don't use microfluidics.
I'm being halfway facetious here.
But this is actually a project that my own lab has undergone,
where we have an anemia test. It was a microfluidic,
but we decided that it would cause too much error.
When you shrink down a microfluidic, a little bubble,
a little piece of dust, that can kill the whole thing.
So, we said, "Forget that.
We're just going to use a bigger chamber."
And we have an anemia test here where,
based on the color of the chemical change,
it correlates with hemoglobin levels.
Sensitivity and specificity is not too bad --
I won't go over it too much.
This was FDA cleared by the U.S. FDA in 2017.
It is now licensed to our company,
and we got prescription home use just last year.
So, sometimes getting rid of the microfluidic is the way to go.
Okay. So analysis is another way we can do things.
So, with the advent of machine learning and AI, we said,
"What if we create some open-source software
to help the field?"
So, anyone with a microfluidic can download our software,
and we can help do automated analysis
and even plug-and-play machine learning algorithms.
Just push of a button, we can help explore it.
So, that's all part of it.
Okay. So, here's my last slide. Where are we?
Well, we actually, unfortunately,
chose to work with a very complex disease --
with few, if any, validated clinical markers,
with clinical endpoints that are difficult to define or quantify.
All of you are involved with clinical trials.
How do you define your pain endpoints?
It's always tricky.
It involves a very complex bio sample -- whole blood.
And heterogeneity, in and of itself,
is a hallmark of the disease. So, where are we headed?
I would say that we're getting there [laughs].
This is my last few slides.
This, in some ways, is just how long the process takes, right?
We have proof of concept at trying to figure out
the right pathway to implementation is never fast.
And we are literally building planes as we fly them.
When I talk to the clinical pathologist, they say,
"Sure, we'll help you refine your device."
"What's the biomarker?" We said, "Oh, we don't have one yet."
Like, "Why are you making something
you don't have a biomarker yet?"
"Well, we're making something to figure out the biomarker."
And they're like, "That's ass backwards."
They're like, "Yeah, I know.
But that is where we are as a field, right?
So more longitudinal studies is needed.
And it may be that single assays may be less clinically useful
than a panel of biomarkers."
We do see some possibilities of the analysis part, right?
Data science tools can probably help us.
And maybe we need to consider that microfluidic assays
may actually not be the path forward.
Maybe the way to best predict vaso-occlusive episodes
is not with whole blood.
Maybe it is an omics-based approach.
Vivian Sheehan just published a nice commentary a few months ago
describing maybe that's a possibility.
Anyway, I would like to end with thanking my lab,
thanking our collaborators, thanking our funding.
Thank you. Okay.
[applause]
Female Speaker: Yeah. Because you will
take questions of each speaker,
since Dr. Alan Docter [phonetic sp]
has to leave after the second talk.
So any questions, anybody?
Manu, I see you get up. Are you going to ask questions?
Male Speaker: I'll wait for the rest.
Female Speaker: Oh, Okay. I see a hand up there.
Scott Peselak: This is Scott.
Sorry. Scott Peselak from UPenn. Really great talk.
I'm just curious if you have thought about ways
to differentiate between cell-intrinsic
and cell-extrinsic aspects
that might affect microfluidic flow in your system
and just in general.
Because, you know, in the patient's hydroxyurea,
obviously, we get increased fetal hemoglobin,
but also modulate a lot of other aspects of whole blood, too.
Wilbur Lam: The answer is yes, and we can talk later.
Scott Peselak: Okay. Sounds good.
Female Speaker: Just one.
Oh, sorry, Mary Lou [phonetic sp]. I'm sorry.
Mary Lou: One thing that has concerned me
with adhesion assays generally in a longitudinal sense
is that I can hypothesize that the most adherent cells --
the most adherent red cells,
will actually kind of disappear from the circulation
during a vaso-occlusive episode because they are adherent.
And so that it would be possible to get really the opposite
of what you expect in these kind of microfluidic assays,
or for that matter, in our less microfluidic assays
that my own lab uses.
And I was just wondering if you have thoughts about that.
Wilbur Lam: Yes, we have. In fact, in some of the studies,
we have actually seen exactly the opposite, right,
where in the acute setting,
we actually see a decrease in adhesion,
and then later on, it goes back to baseline.
And I think I didn't have time to go over it real quick,
but I think we as a field really need to figure out
exactly how we want to use these things, right?
Are we trying to diagnose a vaso-occlusive event?
Are we trying to predict?
Or are we trying to also maybe, if we do it longitudinally,
are these tools to maybe demonstrate resolution
or convalescence of these episodes?
All these are questions that we're dealing with,
and I think it's all about getting more data
and getting as much as we can
because I think we need to have an unbiased approach here
and not be so focused on our hypothesis that,
oh, yeah, adhesion is going
to directly correlate with some event.
It may be, like you said, it may be the complete opposite.
So really trying to take a non-biased approach
to all the data, getting as much data as we can,
I think is the way to go, yeah.
Male Speaker: I was wondering if you've ever used
the microfluidics for selection purposes,
so if you're looking for a particular cell,
whether or not, like, for example,
can you discriminate a sickle red cell from an adult
or even a fetal cell using microfluidics?
Wilbur Lam: Yes. So from the research side,
we have, and, you know, the best way to think about it
is these are physiologic systems
that are easily coupled with microscopy, right,
so staining for whatever subsets.
We've certainly done that.
And, yeah, we've seen a lot of things that we would expect.
You know, reticulocytes tend to be very adhesive in these.
Male Speaker: But are we selecting for possibility?
Wilbur Lam: Well, you could, right?
You could do this downstream selection.
There are fancier fluidics where you can trigger a valve to open
so that you see a population that you want,
and it can go to a certain bucket,
almost like akin to, like, a flow cytometry sorter, right?
There are ways to do that.
And some groups, including ours to some extent,
have done that selection process.
Female Speaker: So, Wilbur,
I have one question before I let you go.
So all these fancy devices that you have,
you all call it vibrant or whatever,
and try to simulate the, you know,
the blood flow through capillaries.
Why can't they do it in vivo, in real time,
just like we use pulse oximetry?
So then you take away all these other complications,
and then there is still some intrapatient variability,
but at least for long-duration flow.
Wilbur Lam: Yeah.
So I don't disagree with you, Sui-Lei [phonetic sp].
I think there will always be a role for in vivo techniques,
and there have been.
So there are, for example, ultrasound-like devices
where you can actually measure flow.
There are oximetry devices now
that can even get a sense of blood flow, even intracranially.
Like you can get a slice of it under,
so for example, Erin Buckley, who's a bioengineer at Emory,
you know, she's done a lot of work
in the sickle cell field as well in this.
Everything has pros and cons, Sui-Lei.
So the cons to what you're talking about,
and we've demonstrated this,
is you take some of those in vivo ones,
you do it on the finger, it gives a readout.
You go to the next finger,
it's a totally different readout, right?
Female Speaker: Even the same person?
Wilbur Lam: Even the same person, right?
So again, we're talking about heterogeneity, right?
Unfortunately, it's a hallmark of the disease.
Every single technology has its own pros and cons.
My own expectation is in the next 10 to 20 years,
we will see a lot more of these things out in the market.
Each will have a specific niche,
and each will have a specific clinical use case
that we might not even be able to predict right now.
Female Speaker: But just tell them to use the same finger,
and don't change to, you know,
the other thing is the nail polish
which they use too, especially for the women.
So I think I'm going to introduce the next --
oh, there's one more question.
Female Speaker: So Wilbur, I'm really excited.
I remember asking you to put those videos in the clinic
while I was at Emory.
Applicability. So these things are really nice, good,
but how well are we using them?
The videos you've got of blood flow,
showing the blood flow of the sickle patient on HU
and the one who's not,
has it changed your HU compliance?
Wilbur Lam: We'll have to ask our colleagues that.
I don't know, actually. I mean, we use it in the clinic,
and I do say, at least anecdotally,
when I see a patient and I show that to them.
So, I've actually had a couple of parents actually say,
"Oh, I heard you got this video, can you show it to my kid?"
And yeah, what's nice about it
is that every kid has a phone now,
so I just, you know, give it to them.
So they see it on a platform that they're used to.
I anecdotally have personally at least gotten positive feedback
from at least a handful of kids where they're, eh,
and then they see it and they're like,
you know, I'm going to show my age, right?
For those of you who remember the, "This is your brain,
this is your brain on drugs," right?
So I use that type of analogy, all right?
This is your blood not on hydria,
this is your blood hydria, and they're like,
"All right, maybe I should do that, yeah."
Female Speaker: I'm chuckling because when you told a story
about your student picking up your driver's license,
it's like me, you know, is refused our age, too.
We used to keep our lab books, you know, books.
So she looked at the book, she said, "Oh my God."
I said, "Yes, before you were born."
[laughter]
Okay, this is what David Van Roy used to tell me:
"Before you were born."
So now I tell it to the students.
Okay, so now time's up. Yes, no, no. Yes, absolutely.
Okay, so I have great pleasure to introduce Dr. Alan Docter,
who actually we have not met,
but I heard his presentation and one of our extramural,
you know, talks that were given in the NHLBI.
And I thought, "Wow, this is so relevant to us."
So Dr. Alan Docter is professor of pediatrics
and bioengineering at the University of Maryland
School of Medicine. And he's going to talk to us
about the role of red blood cell ENOS
in optimizing red cell phenotype
during circulation. Thank you so much.
[applause]
Alan Docter: Thank you, Sui-Lei,
and thanks for the opportunity to show this work to you all.
It's going to be a bit different
than the other talks that you've heard.
And to confess, I'm not a sickle cell specialist,
not even a hematologist. I'm an intensivist.
But so what is the point, really,
and why is this of interest?
So the principal task for red blood cells
is to move oxygen from lungs to respiring cells.
That's the point.
And they're within a delivery system
that includes the heart and lungs and vascular tree.
And it's not just that.
The point is to be able to adapt.
So right now, your oxygen consumption is not so great,
especially after eating a little maybe, you know,
but if you get up and say there was a fire alarm
and we had to go down the stairs,
you have to suddenly increase your oxygen consumption.
So the system has to be adaptive.
And in fact, when we evolved,
there was a sort of a 400-million-year forward
genetic screen evolution, right,
which favored people who could run away from tigers very fast
and rapidly scale their oxygen delivery.
And so red cells have a big part in that.
It's not commonly appreciated.
So one of the systems I'm going to talk about is this ENOS.
For those that don't know what ENOS is,
that's an enzyme that makes nitric oxide.
Most people have heard of nitric oxide.
Nitric oxide is a very important signaling molecule
that coordinates oxygen physiology.
And so red cells dispense this molecule in our vascular tree
in a way that supports oxygen delivery homeostasis
under stress. And that's really important.
Turns out that sickle red cells don't do that so well,
even when they're not sickling.
And so we are studying red cells,
which are surprisingly poorly understood,
and identifying some new phenotype features
that are relevant to oxygen delivery homeostasis
and particularly relevant to sickle cell disease.
And so that's what I'm going to talk to you about.
So this may be a new drug target.
These are my disclosures.
There's nothing important or relevant
to what I'm talking about here today.
And so the oxygen delivery system
that I'm talking about is this cascade,
really, of, you know, interlinked systems
that coordinate compensation for coupling
between oxygen delivery and consumption when it changes.
So, it might go up and you need to deliver more,
or the availability might go down,
like you have asthma or sleep apnea or pneumonia,
or you go to altitude,
and, or you may have a lower cardiac output.
Whatever it is, you still have to balance
the delivery and consumption or we've got problem.
And those problems are quite obvious
when you're taking care of patients
with sickle cell disease.
Red cells are at two points in the system,
capturing the oxygen in the lung and releasing it in tissue.
Those are paradoxically opposites, right?
So the same cell has to do one thing in the lung
and another thing in your muscle and do it effectively and well.
Why? How does that happen? And it turns out that red cells
are actually pretty sophisticated,
as I'll point out.
So, this is a study that was done quite a while ago
asking this simple question, right?
So what governs blood flow distribution?
It wasn't really understood. Nobody got it.
It's not like we're studying what governs cardiac output
or what governs how to stabilize blood pressure.
What governs blood flow routing? Okay.
So right now you're routing blood
through about a tenth of your potential sort of capillaries.
We are really not perfusing most of what we can perfuse.
And everybody that's exercised or gone to the gym
and you see the people who take their pictures
and they're all pumped up, right?
That's because there's an increase in blood flow.
It's not the muscle.
It's the muscles are engorged with blood.
That's because you can suddenly really scale
by logs how much blood flow goes.
So why? Nobody figured. Nobody really understood that.
And rather famous physiologist,
for those of you who may have heard of Guyton,
right, asked this question, simple question,
and there was a big idea back in the '50s
that this was the autonomic nervous system
that nerves somehow opened and closed blood vessels.
And so he did something that we can't really do
to companion animals anymore. He had this spinal preparation.
So, he decoupled the autonomic nervous system
from the periphery,
and he isolated the femoral artery
and the femoral vein from a dog
and did a really simple, beautiful experiment.
He put a flask of blood above the femoral artery
and measured how fast it took for it to empty.
That's it.
And then he did that with blood
that was progressively less well oxygenated.
You can see the x-axis there on the left starts with 100
and goes to zero.
So, that's 100 percent saturation of the oxygen
in the blood on the top, and then to zero.
And the y-axis is how fast the flow rate,
how fast did it run out.
You can see the blood ran out. That's relative.
So the blood ran out like five times,
three and a half -- sorry --
three and a half times faster with fully desaturated blood
than fully saturated blood.
Now, how's that possible? You can ask Wilbur.
He'll tell you the blood's not that --
it's not less viscous, so to speak.
It's not becoming easier to go.
It's actually the impedance to flow in the circulatory system
is changing as a function of how well oxygenated the blood is.
And it turns out if you look at the bottom, same x-axis,
y-axis shows oxygen delivery, which is content times flow.
The flow is increasing just enough to stabilize
the O2 delivery.
And then it falls off in a linear fashion around the time
where we start to see problems in the hospital
where we can't compensate for hypoxia,
saturations around 70 or so.
So, he wrote in a prescient way, "It's possible that red cells
prevent accumulation of inhibitor or release a factor
that enhances vascular responsiveness."
Pretty clever guy.
Took a while to figure out what that was.
So, only recently do we understand
that red cells monitor oxygen gradients
and either dispense or sequester nitric oxide in response
to those gradients to match impedance to flow to demand.
And so red cells entering a very well-perfused blood vessel
will sequester this vasodilator,
and they leave behind a slightly smaller vessel than they enter.
And red cells that enter a hypoxic blood vessel
add the amount of nitric oxide,
and they leave behind a slightly bigger vessel.
This is happening on a millisecond time scale,
with trillions of red cells all like itty bitty little IO chips
flying around your circulation asking, "Is there enough oxygen?
Is there enough? Is there enough?
You want a little more? You have too much?"
So every red cell is asking that question
and adjusting the blood vessel size.
And that doesn't happen so well with sickle red cells.
I'll explain why.
So, the adaptive change. I told you about this already.
Okay. Red cell phenotype is adapting,
and it turns out that the phenotype is oscillating
during circulatory transit.
So, red cells that are going through arteries
are different than the red cells that are returning in veins.
They have different oxygen affinity,
different deformability, different chemistry,
different energy metabolism.
And in fact, they're doing this in proportion
to the amount of oxygen lack or availability.
And so that is happening on a subacute time scale
or acute time scale during circulatory transit,
which is what I'll talk about today.
Subacute time scale, which is like high altitude adaptation --
everybody knows red cells change during that.
They can also change with anemia, sustained anemia,
or stress, or increased odor consumption,
and then a chronic time scale
where the actual transcriptional phenotype
is part of the adaptive change.
So, the failure of red cell phenotype adaptation
contributes to morbidity and non-hematologic disease.
That's newly understood,
but it's also an underappreciated part
of hematologic disease as in sickle cell.
And here we're going to focus.
So mature red cells harbor a functional ENOS.
Without getting into those details,
a lot of people think mature red cells,
like, they forget their cells.
Like it's just an envelope of hemoglobin,
and when we talk about it, we just say, like,
"What's the hemoglobin?" Right?
Nobody is asking, like, well, how well does it work?
Like in what other disease, what other specialty?
You don't see cardiologists counting myocytes, right?
Or a nephrologist counting nephrons.
They like, "How well does it work?
Like, I'm sorry,
but hematologists like count the cells,
but don't always ask how well they work.
And that's an important distinction."
And people forget they actually are really complex,
as I'll show you.
These are mature red cells that have this signaling in it.
And people's like, "That can't be true."
And so it was a big revolution back in the day or 10 years ago
or so when people said,
"Oh, this is going on in mature red cells."
So, what I'm going to do is show you --
go through this kind of fast --
some of them like proof that red cells
are doing this sophisticated signaling,
and then explain what it does
and how it helps support oxygen delivery,
and then show you a bit about why it doesn't work in human,
in sickle cell disease.
So, this is an assay that's reporting the amount
of nitric oxide being generated inside a red cell.
And first of all, that red cells even do that was new,
but it turns out that red cells do that
as a response to how much oxygen they have.
So this is on the y-axis, is time on the right.
Left y-axis, on the x-axis is time.
On the right y-axis is hemoglobin oxygen content.
The left is the amount of NO being produced.
And you can see that in the circles,
the red cells are, have oxygenated.
The squares, they're deoxygenated.
And when you deoxygenate the red cell,
it makes more nitric oxide. Okay.
Turns out that if you take red cells from mice
that don't have nitric oxide synthase, this doesn't happen.
Okay. That kind of makes sense,
but people used to think it was nitric oxide
that was getting captured from endothelial cells
and then processed in red cells.
In fact, there was a Nobel prize for that wrong idea,
but red cells actually make their own.
So how does that work?
Like, how does the enzyme get turned on?
So, we started thinking about calcium and calcium
signaling activates nitric oxide synthase in other cells.
Does calcium signaling work like this in red cells?
And red cells have a channel called Piezo1,
which is, opens mechanically.
So here, without going into the details,
we're looking at how much calcium is produced
with the same construct.
Okay. We're deoxygenating the cells.
And blue is deoxygenated, red is oxygenated,
and open squares, we've taken away all the calcium.
You can see that deoxygenated red cells
have more calcium coming in.
And if we block this specific channel with the GSM TX,
you don't get it.
So Piezo1 is opening in red cells,
and Piezo1 is bringing in calcium
when red cells deoxygenate.
So, there's a problem there that I'm going to have to deal with
because Piezo1 is only supposed to open when cells deform,
and it's a mechanical channel.
So, how is it opening when red cells are just sitting still?
But we'll talk about that.
Now, the other thing that's interesting is that
the ENOS, this enzyme, is flying around inside the red cells.
It's moving from the cytoplasm out to the periphery.
And of course, we're showing here
there's none in the ENOS-deficient red cells.
On the left under red is this enzyme in oxygenated red cells,
and on the right under blue, it's in deoxygenated red cells.
You see it's moving to the membrane,
and that's also kind of strange.
This is migrating back and forth inside red cells,
and how could that be happening? And really, to really be sure,
these are optical tomography sessions through red cells.
You can see it's very clearly dispersed and not.
And these are the mouse cells.
So it's definitely happening. And this is further --
we're quantifying the fluorescence --
and you can see that difference.
And if you prevent the conformational change
that red cells make when they deoxygenate,
you prevent that change.
So it's actually beginning with this conformational change.
It's not the amount of oxygen.
So also, if you open the calcium channel
without deoxygenating the red cell,
you can make nitric oxide move.
So, it's a calcium signal that hemoglobin is deoxygenating
and causing the calcium to come in.
So then, what's next? There's some phosphorylation.
So ENOS has complex regulation,
and it is getting phosphorylated in a particular pattern
in deoxygenated red cells that promotes activation.
And that pattern can be recapitulated
with all the signaling that I'm talking about.
You deoxygenate the red cell, you open the calcium channel,
you prevent the hemoglobin conformation change,
you can cause the correct pattern.
So, we see that the hemoglobin conformation is changing,
calcium is coming in,
ENOS is migrating, it's getting phosphorylated
and producing nitric oxide. And again, we can produce --
this is just data confirming again in a different way --
that we can not only recapitulate
the phosphorylation pattern,
we can recapitulate the amount of nitric oxide being produced.
So the real question is,
how's this all getting started in the first place?
So I'm like, how does a mechanical channel open
without the cell being deformed?
Well, it turns out red cells have myosin,
and there's a special form called non-muscle myosin
that we wondered,
"Maybe this is pulling the Piezo1 channel open."
It turns out if you block this protein, non-muscle myosin,
then you can prevent the deoxygenation-induced
opening of the calcium channel
and the production of nitric oxide.
So, what's going on is hemoglobin conformation
is changing,
it's docking on a special protein
in the membrane that's activating non-muscle myosin.
Non-muscle myosin is pulling open a calcium channel.
Calcium is coming into the cell, causing nitric oxide to migrate
and get activated and then produce nitric oxide.
That's a lot for an envelope with hemoglobin.
So then, what happens?
What happens after the nitric oxide is made?
It signals ubiquitously in a system
called protein S-nitrosylation, which is like phosphorylation.
We looked at this, and this pattern --
the heat map -- you can see these are membrane on the left,
the cytoplasm on the right,
deoxygenated red cells in the left panel,
left sort of column, and oxygenated on the right.
You can see that the pattern of NO nitric oxide on protein
is totally different. These are thousands of proteins
that are changing as a function of nitric oxide being activated.
So the red cell phenotype is being manipulated
in a very sophisticated way
as the red cells go through circulation.
And this is the glycolysis and other features.
And this is just showing, you know, you just don't see
that when you don't have nitric oxide enzyme around.
So what's the consequence?
So first thing is the way red cells use energy changes.
Red cells, ironically, don't use oxygen, right?
So, they have no mitochondria, but they burn glucose.
They burn glucose anaerobically.
And the way they route the glucose changes.
And glucose is routed more towards antioxidant systems
in deoxygenating red cells,
primarily because of this nitric oxide signaling,
so that they can recycle NADPH and glutathione,
which is very necessary for the thiol-based antioxidant systems.
That makes perfect sense.
And then, when red cells come back around, okay,
then glucose is routed in a different way,
and you're making NADH and ATP,
which is reducing methemoglobin and governing ion channel flux.
So, deformability.
The red cell cytoskeleton is very, very well characterized
but has principally been thought of as a static system --
that it's not that adaptive.
Now, take a neutrophil or a macrophage
that is sort of moving around like this.
I mean, you've got incredible versatility in the cytoskeleton.
The cytoskeleton can be a motor,
and red cell deformability is changing
during circulatory transit.
But what's happening with the cytoskeleton?
Well, it turns out the cytoskeleton is assembling
and disassembling every few seconds
as red cells traverse our circulatory tree.
This is on the top.
We're looking at actin at the bottom.
We're looking at spectrin.
You can see that there's a diffuse cytoskeleton
that's complex going throughout the cytosol
when the red cells are oxygenated,
and it collapses on deoxygenation.
This is happening over and over again.
Turns out that doesn't happen
if you don't have nitric oxide or nitric oxide synthase.
And you don't have that if you prevent
this whole signaling system that I was talking about before.
So, what does that mean for deformability?
And so we had to figure out a way to measure deformability
without deforming the cell. So, if you deform the cell,
then you open the calcium channel,
and now you've got a problem.
So, we're using something called Brillouin spectroscopy,
which is a no-touch way of measuring deformability
through photoacoustics that is quite interesting.
But the bottom line is that red cells --
and this is just validating it
with opening the calcium channel causing deformability
and stiffening the red cell with something called diamide
and showing that we can measure this.
It turns out that when you deoxygenate the red cells,
they have an increase in deformability
because the cytoskeleton is collapsing.
And this doesn't happen
if you don't have nitric oxide synthase.
Just in the interest of time, also the Bohr effect,
which is a complex control of pH within red cells,
fails when you don't have nitric oxide synthase.
And that's necessary for O2 delivery homeostasis.
And the vasodilation that I told you about
before that was the basis of the Guyton experiment
also fails when you don't have nitric oxide synthase.
And if you try to run mice that don't have nitric oxide
synthase in their red cells, they fall over.
So, basically, you need it to scale your production.
Now, what happens in sickle red cells?
So, we took a look,
and it turns out the regulatory controls of the system
don't work in sickle red cells. So, the sickle red cells,
which are supposed to have an increase in calcium signaling
when you open Piezo or when you close Piezol,
are insensitive to manipulation of the Piezo1 channel.
So there's some other calcium flux going on that
is interfering with this system.
And therefore, the nitric oxide synthase
is not responding to normal controls,
and the regulation of nitric oxide synthase --
the phosphorylation pattern -- is abnormal.
It's actually turned on all the time, which was a surprise.
And it turns out the cytoskeleton doesn't collapse
during circulatory transit the way it should.
And because it doesn't collapse during circulatory transit --
you can see these are red cells where spectrin and actin
is not disappearing when red cells deoxygenate.
These red cells, when we examine them with Brillouin microscopy,
don't show the deoxygenation-induced
increase in deformability.
In fact, as you all know, it goes the other way.
So this is just the same thing with sickled red cells.
So, it turns out that ENOS is coordinating
a phenotype modulation during circulatory transit
that supports oxygen delivery homeostasis
whenever there's stress.
And that means, you know, we, okay,
if you have normal red cells,
then you can run up and downstairs and not fall over.
You can run up and downstairs without getting claudication,
and you can have a little asthma,
and you don't have a stroke,
and you don't have all these problems
with oxygen delivery in the periphery
that fail every time there's a little bit of change
in oxygen delivery availability or consumption.
And that this is a complex system
with a lot of potential druggable targets in it.
So, I apologize for going through
a lot of that kind of fast,
but I wanted to give you an overlay of an interesting system
that may improve our understanding
of why patients with sickle cell disease
can't seem to adapt to hypoxia
in the way that a normal person can.
And it's not just about hemoglobin polymerization.
There's much more to the disease than that.
The sickle red cells just are metabolically different
even when they're not sickling.
So, I want to acknowledge the people that did all this work --
principally, the two in red there in the lab
who did all of the work, in fact,
and the many colleagues and support for the program.
Happy to take any questions.
[applause]
Female Speaker: Thank you so much.
It was a really, for me, you know, a real eye opener.
Actually, Manu has got his hand up.
Manu Platt: Great. Thank you, Manu Platt, NIH.
This was interesting.
So, I'm talking next, but E, as you know,
the E in ENOS is endothelial, right?
Because I come out of a shear stress
endothelial cell biology background
where we think it is one of the genes quite unique
to endothelial cells.
So, all of this is blowing to me,
and I've got to dig into the literature you showed us.
But one of my things that we know in endothelial cells
is ENOS is shear stress-regulated
so that it gets phosphorylated under high flow and high shear.
Endothelial cells being adhered cells, so you can --
they are standing still while the blood is moving over them.
How do you imagine that looks in your RBCs
that are experiencing shear
while they're moving with the blood flow,
but some of your activations and localization
from internal to the cell to the cell membrane?
And I'm just curious how that plays
with these RBCs under flow.
Alan Docter: Yeah, great question.
And so obviously what we're doing here is trying to --
we were trying to dissect or decouple something
that's going on at the same time in real life.
So as the red cells are deoxygenating and reoxygenating,
they're also deforming. And the deformation is also --
the intensity of the deformation is linked in part, obviously,
to the oxygen gradient in that particular spot.
So, the -- I would think the deformation signal
would be a change in tandem with the deoxygenation signal.
Now, we haven't begun to look at that yet.
We're only really been looking
at what's happening with deoxygenation
and how Piezo is opening just with deoxygenation,
which we thought was kind of amazing
that there's a motor protein that's kind of tickling it open
when the cells are sitting still.
So, when we found that when we were using, say,
an instrument that has --
say, you're familiar with the Lorca instrument, right?
Which, when you try to deform,
when you are measuring deformability
by deforming the cell,
we're interfering with all this signaling.
So my suspicion is that these things are acting in synergy,
but we haven't begun to evaluate it.
But yes, good question.
And the E, you know, it's a holdover.
It's really in more tissue types than just the endothelium.
Female Speaker: That was remarkable.
Thank you.
What do you think the impact of medications
that delay conformational change would have in this system?
Alan Docter: Yeah, great question, right?
Okay. So if you, so you might initially think,
"Oh, that's going to be a problem," right?
Because we need that signal in order to activate the program.
This is really about 10 percent or less of the hemoglobin it,
so most of the hemoglobin is nowhere near the membrane.
There's a special population of hemoglobin
that's involved in this type of regulation
that's in the submembrane domain,
and it's the first hemoglobin to deoxygenate.
So what -- there is an interesting literature
on the cascade of how much --
where in the red cell is the hemoglobin,
is the oxygen actually coming from,
and where the conformation change is occurring.
So, there's a risk that drugs like Voxelotor
or others that shift the O2,
you know, affinity would be retarding the signaling.
But we haven't even begun to explore that.
There are ways to compensate for that, perhaps,
but we haven't -- that's a good question and probably relevant.
Male Speaker: Well, I was wondering,
I was wondering, have you done a comparison
between F cells and adult cells and their ENOS content?
Alan Docter: Yes, a long time ago,
but not here. So we did it a long time ago
when we were looking at sort of crude --
the amount of nitric oxide that can be generated
to dilate a blood vessel -- and F cells generate more.
So, I don't know why. And that's funny because
they have a right-shifted oxygen dissociation curve,
but they generate more NO than a non-F cell.
I don't know why, but we haven't looked at those cells
with this particular system.
Male Speaker: Yeah. One thing that interests me is how,
in patients who could easily discriminate F cell content,
and I'm just wondering if this is a way of doing
just that without having using antibody.
I mean, can you just look at a content of ENOS
or something else just to see?
Alan Docter: Yeah, possibly. I don't know that.
Female Speaker: Yeah.
So time's up, but Marilyn [phonetic sp],
you've been dying to ask a question.
I'll let you have the last question before Dr. Dorff
[phonetic sp].
Female Speaker: So when patients are treated with Voxelotor,
their deformability of their red cells
as measured by Lorca improves,
whereas it's reduced for a lot of sickle cell patients.
Is there a technically feasible way to measure
to what degree deformability is helped or decreased
by the degree of kind of reversible polymerization
of hemoglobin in the cell?
Or, for that matter, what percent of hemoglobin
is proximal to the membrane,
and what percent is it so that you could say
what the percent contribution is of what you've just
very beautifully presented versus kind of --
I will call the traditional,
you know, it's all about the hemoglobin?
Alan Docter: Yeah, yeah, yeah.
Well, there's a lot to unpack in there,
but there is a technique that is in development now
that involves something called hyperspectral imaging,
which can monitor the degree of protein polymerization
and whether -- and also whether --
that is cytoskeletal or hemoglobin.
And it's not, you know, it's not --
how can I put it, to use Wilbur's language,
it's like TRL-2, so not ready to be deployed by any stretch.
But there -- and it can go through the skin.
So there are, there may be approaches to monitoring
the degree of hemoglobin polymerization
in a transcutaneous way,
but it's a few years -- more than a few years away.
Female Speaker: Thank you so much.
So I will let you go to give your other talk,
and I'll see you later.
I'll let you go, and I will be back.
Alan Docter: [inaudible]
Female Speaker: So, yeah.
[applause]
So, it's my pleasure to introduce Manu,
who I kind of know very well and work with.
So Manu is the inaugural director of the NIH White Center
for Biomedical Engineering Technology Acceleration,
which is housed within the National Institute of Bioimaging
and Bioengineering. Did I get it right?
So, Manu, thank you very much. So as I said, 25 minutes.
I know you've got more than that.
Manu Platt: I've got my timer.
Okay. I'm really looking forward to this.
Female Speaker: Thank you, Manu.
Manu Platt: Great. Thank you all very much for having me.
And so many great people here in the room
between today and tomorrow
whose work I've been building on and reading for years.
I'm quite excited to share my work with you all.
So, as mentioned, I am at NIH at the NIBIB,
mentioned by Wilbur, but I just moved there about 18 months ago.
I started my career at Georgia Tech Emory
in their Department of Biomedical Engineering
and actually got my PhD at Georgia Tech Emory
in cardiovascular endothelial cell and shear stress biology.
That then we shifted to sickle cell
when I started my own independent lab.
But I always, as the director of this beta center,
I do have to plug it everywhere I go because my job,
one of them, is to get people in the extramural world
to be interested in working and partnering with people
in the intramural research program at NIH.
But one of the other goals that we're doing
is to allow the other intramural researchers at NIH
to access engineering tools, analytical methods,
computational biology, biomaterials, microfabrication.
So, again, also always advertising that we are building
these internal capabilities at the NIH,
but again, open to working with others.
There's my wonderful deputy director
who handles things when I'm gone.
But again, one of the other things that we do is, again,
to help build these relationships.
We have a huge event coming up at NIH on October 22nd,
building bridges across NIH
and with the broader engineering community,
where we have an intramural plenary talk
via Dr. John Tisdale [phonetic sp]
here and an extramural plenary talk by Dr. Rashid Bashir
[phonetic sp].
And there's multiple talks happening on campus.
We'll be taking over the campus,
networking, bringing things together
so the engineers can meet with the clinicians and researchers.
So if you'll be around Bethesda, free registration
because your federal taxes go towards our support.
So, thank you. And we were fortunate
that Dr. Monica Bertagnolli [phonetic sp]
will do a welcome for us at the beginning.
But I do have a research lab. My lab did move to NIH as well
and the NIBIB intramural program.
A major focus of my work is pediatric strokes
and sickle cell disease and some of the underlying mechanisms,
which I'll be talking to you about today.
And we use a number of different techniques to assess this,
and I'll talk about a few of those.
So, I say my lab studies tissue remodeling.
And so, just to orient people,
what I think tissue remodeling is,
I look at it as how do you go from this diseased vessel
this healthy vessel on the left
to this diseased vessel on the right.
So that is a human coronary artery,
and you can see this cross-section.
So blood is coming out of the board,
and it's nice and happy and open --
minimally diseased, we would say.
But you can see for that atherosclerotic plaque to form,
where just that little slick of white space
is where the blood would now be moving.
That huge plaque has remodeled that artery wall.
And so some of the initial things that happen
there are degradation of some of the structural proteins
that make up the artery wall -- the elastin and the collagen --
that leads to cells receiving different cues.
They begin to migrate, infiltrate that area,
deposit new proteins,
and there's a feedback loop that continues.
So, what is happening? But what's important is not only
is the biochemical environment of that artery wall changing,
but the biomechanics change.
So I can appreciate Dr. Theon's [phonetic sp]
opening about considering the mechanics and the biology,
which is something that,
as biomedical engineers love to bring together.
Another thing that people understand is from osteoporosis.
You go from this normal, dense, mineralized bone matrix to this.
You lose some of the mineral calcium.
Again, loss of the biochemistry changes the biomechanics,
and you see this predominantly in postmenopausal women --
greater risk of bone breaks, right?
But we also know in sickle cell, the sickle bone disease
that also takes into account this bone remodeling.
So really what my lab studies is how things fall apart, okay?
And so we can understand those mechanisms
so we can stop it and hopefully keep it together
because some of these structures aren't easily regenerated
or repaired, okay?
But these agents of destruction that I really focus on
are these enzymes called the cysteine cathepsins.
So, they were first identified in lysosomes,
where they play key roles in protein turnover.
But they're upregulated in disease
and actually get released and secreted,
where they can do damage to a lot of matrix proteins.
Again, first there were we began learning about them
in cardiovascular disease and atherosclerosis,
where endothelial cells begin to express them
under different fluid environments.
Also, the smooth muscle cells can express them
as they migrate and respond to inflammatory cues.
All of this together begins to remodel the milieu
of the artery wall.
And so also where mechanics come into play are also blood flow.
So we think about, I say, hemodynamics,
and I think some hematologists do it differently.
So, I mean fluid shear stress
and the mechanical force of blood
as it moves across the blood vessel wall.
So, people knew for years, back in the '50s,
they had identified that were atherosclerotic plaques
were forming word regions of bifurcations
or sharp turns in the vascular tree.
So, a pathologist called the aerospace engineer buddy,
and they were like,
"Oh, of course they're happening here."
There are these regions of flow separation.
There's not a laser thing on here right now,
but they did the regions of flow separation.
I'm sorry, regions of bifurcations,
you get flow separation, flow reversal,
and particularly with the cardiac cycle of systole
and diastole.
You can actually get blood moving backwards
depending on the geometries of the artery wall.
And you can see here where you see
these atherosclerotic plaques forming --
sorry actually correlates with where you have the regions
of flow reversal in the artery wall.
And so, my PhD was all about learning
how the mechanical forces alone drive gene
and protein expression, which really blew my mind.
From my undergrad degree was biology,
where in biology, I think you learn what happens, right?
This binds to this, this does this.
But what I really appreciate about biomedical engineering
is it told us more about why it's happening.
Ourselves and our tissues obey the same mechanical laws
that, like, a brick does, right?
They also fall with gravitational acceleration.
So, all of those things are happening inside of cells.
And so that's where we found out that the system could have
things that I mentioned that are powerful collagenases
and elastases are actually turned up at sites of disturbed
flow where you see plaques and vascular remodeling,
and turned down at the straighter parts
where you get high unidirectional blood
flow or unidirectional shear stress.
So, these are mechano-sensitive proteins
that are regulated based on blood flow.
Fast forward from then
to when I started my independent lab in 2009
and really began to have some mentors.
I was back in Atlanta
and started caring more about sickle cell disease,
which we all know these facts,
so I don't have to state them for you all.
But what really blew my mind when I heard that children
with sickle cell disease were having strokes.
I had been studying cardiovascular disease
and atherosclerosis,
and we think about that happening when people are 50,
60, 70, 80 years of age --
which was already terrible -- but kids having strokes?
Nobody was talking about this about after a world,
and then again the complication here,
I mean, at the complication,
the terrible part is 11 percent of these children
will have a major stroke, meaning there's some paralysis.
But then when you find out
that a third will have a silent stroke,
where it's occurring in a cognitive part of the brain
that isn't presenting, kids -- this makes no sense to me --
and understanding that the red cell sickle
under deoxygenated conditions, but strokes are happening
in the oxygenated vascular conduits.
So, it really broke a lot of the themes,
and paradoxes started to really grow
for how we would think about it.
But some great work, again from people in this room
and others, showed that --I'm going to hypothesize about
what these mechanisms could look like --
and they were shown to be lasting in collagen degradation
in the artery walls from autopsy specimens.
A lot of hypotheses, again, you know, scavenging,
high shear, clotting,
and a number of different hypotheses
about what could be happening.
But when I also read about this STOP study
by Bob Adams at Medical University South Carolina,
blew my mind because things that I had learned
in my engineering training was --
they said, well, increase that --
increase velocities in the cerebral arteries
a, middle cerebral ACA,
and the ICA, which is the carotid artery.
Those are the children at greatest risk of having strokes.
So, the STOP trial,
they put them on the placebo or blood transfusions
--one of those trials that had to be stopped early
because it was clear those getting blood transfusions
were successful and the others weren't.
Well, I had learned in my fluid dynamics class
that where you get increased velocities,
there could be some narrowing of the lumen
in that location or some other downstream
or upstream differences. And we began to question,
well, we know that these red cells in their white blood cells
and sickle cell are quite adherent.
So, are there some local transient accumulations
and aggregations of red cells?
Again, Wilbur showed those microfluidic images
of the cells coating the walls of those vessels,
even though the blood was still moving.
Or is there some luminal narrowing
where the tissue itself was being remodeled to close?
That would be more traditional of an atherosclerotic plaque
but without the lipid core, obviously.
But, well, either way, where you get that increased blood flow,
downstream of that you get disturbed blood flow
and flow reversal,
which then activates these artery remodeling programs.
And again, we used to think in atherosclerosis --
branches, bifurcation, sharp curves.
But if you have regions
of accumulated red cells throughout,
even the straight parts of the artery wall,
are you now having local pockets of disturbed
flow that could be exacerbating this remodeling
while we're seeing such accelerated artery damage
in people with sickle cell?
So these are the questions we began to tackle.
And Russell [phonetic sp],
where actually met with the Wilbur several years ago,
actually sent us some magnetic resonance angiograms of children
with sickle cell so we could actually take the MRIs,
we could reconstruct the images in the computer
and reconstruct the vasculature,
and then apply computational fluid dynamics
to simulate how the blood flow looks through this.
Because our question was, why is the blood velocity increasing,
especially in certain MRIs?
You don't even see a stenosis at the site
where the increased velocity was being measured.
More paradoxes.
And so, in doing that, the first thing my student found
when he first reconstructed these arteries --
which I was really mad at him at first --
the SS arteries are always way more bumpier than the AA.
And I was like, "Chris, what are you doing?
Are you not doing the algorithms the same?"
Like, he was like, "Dr. Platt, I'm following it
all the way I'm supposed to. I've talked to our expert."
And so, this became really important later
when we put through the same inlet flow velocity profile
in the internal carotid artery,
and just the geometries of the individuals with sickle cell
led to increased centerline velocities
above that 200 centimeters per second target,
which we were shocked. Again, the same inlet velocity --
we had asked him for the patient's inlet flow.
They did vary. But if we assume the same,
we saw these increased velocities
showing just the geometry was changing it.
That led us back to those bumps again,
"Oh, maybe this is indicative of something
because the MRI is like the negative
of the blood vessel, right? It's a space filling,
which could be some of these aggregations, et cetera."
And if we looked at flow recirculation,
which, again, is another indicator of disturbed blood
flow that would cause those altered remodeling pathways.
We could see those readings of flow recirculation
in the cerebral arteries of the sickle cell individuals
but not in wild type,
again with the same input fluid velocity profiles.
We weren't biasing it there.
Again, so that's where we began to develop
this kind of multi-scale approach
of how does the fluid shear stress --
the mechanical forces of blood flow --
influence the gene and protein expression
that caused the cells to express these molecules
that will destroy the artery wall.
So, we worked with the Sickle Cell Foundation of Georgia
to ice or to collect human blood from them --
which now we're going to start working with sweet labor --
quite excited about. And again,
I come from an endothelial cell background and atherosclerosis.
The monocytes bind to the endothelial cell
and the field is lining the wall under sites of disturbed flow,
and then they could enter the wall.
Well, we know with sickle cell there's increased monocytosis
that's happening, and there's increase in inflammation
and TNF-alpha circling in the plasma.
All of these things that could accelerate
this positive feed-forward
to get monocyte binding and artery wall remodeling.
Love to show this picture.
This gentleman on the left is Phil Keegan.
He was one of my first grad students
who really led this work off,
and that's me with the lab technicians
at the Sickle Cell Foundation of Georgia
who collected the blood for us.
But I'm going to zoom through this a bit.
But we found that, of course, with SS PBMCs,
there was greater adhesion to the endothelial cells.
We were doing this with human aortic endothelial cells.
You all in this room had shown this
before we did, so nothing new.
We did ELISA to measure the TNF-alpha levels
in the plasma -- elevated in the SS individuals.
But what we looked for was what were the protease activities
that was turned on after the monocytes
bound to the endothelium.
There's this assay we developed in my group
called that zymography,
where you see increased intensity of white bands --
that's where there's increased amount of the active protease.
Okay And what really --
we really thought was going to happen to it,
that we knew that TNF-alpha would turn on cathepsin K.
The cathepsins are denoted by letters,
so each letter is a different gene product.
Cathepsin K is the most powerful human collagenase
and a very powerful elastase.
In fact, cathepsin K is used by osteoclasts for bone resorption.
Okay, so we're seeing this being expressed by endothelial cells
when they're stimulated with TNF-alpha.
If we co-incubated the cells with PBMCs isolated from people
with sickle cell, again, you get greater adhesion,
and they turn on cathepsin K themselves,
even without pre-stimulation with TNF-alpha.
So, again, there's this revved-up positive feedback
that's ready to happen --
greater monocytes, greater adhesion,
and when they'd adhere, they turn on this protease
to remodel that artery wall.
Cathepsin inhibitors have been sought after by big pharma.
There have been about 16 cathepsin inhibitors
that have made it to human clinical trials.
Sadly, 16 that have failed.
But the last eight -- it wasn't because of efficacy.
They actually were efficacious;
it was side effects that had made them not able to be used.
So, we wanted to look at other signaling pathways
that we could use to reduce
the expression of these cysteine cathepsins.
And in other studies, we found that JNK
and the C-gene signaling cascade --
which we know is downstream of TNF --
but also was able to turn on cysteine cathepsins,
such that, with a JNK inhibitor,
we could reduce cathepsin K activity
and cathepsin V by about 50 percent.
So, you're going to see me come back
to this JNK inhibition strategy.
Okay, so what can we do with this flow information?
Well, we can also recreate these flow environments in our lab
using a cone and plate shear stress apparatus,
where we can grow the endothelial cells
at the bottom of a 10-centimeter dish,
put this cone on top,
and independently program a fluid profile
to mimic what it would look like
in certain parts of the vascular tree.
And, again, very distinctly study the mechanical influences
that drive gene and protein expression.
And so, the endothelial cells --
they elongate and align in the direction of flow,
so you know that they're healthy, of course,
and that your flow regimes are correct.
And this is one of those big things that --
one of the things that I love about putting mechanics
with biochemistry is that there's this interplay
between the two that must be considered.
So, as you see it, we grew the cells under no flow
without any cytokine stimulation.
You get cathepsin V expressed.
You increase -- with incubation with TNF-alpha --
cathepsin K is turned on. I'd shown you that earlier.
If we put that under our pro-remodeling flow,
this is what we see. At sites of disturbed flow,
cathepsin K is already turned on a bit, right,
because it is a shear stress-regulated gene itself.
With TNF, it turns up even more. However, if they are put under
a vasoprotective shear stress profile --
which you would see in the straight parts of the artery
--vehicle, no cathepsin K.
But even in the presence of TNF, under that fluid shear regime,
cathepsin K is not turned on. This is crazy, right?
The same cytokines of -- your biologists are like,
"Well, the signals bind at the receptor,
and all these things should be turning it on."
But the flow is turning it off.
And so, we began to investigate those mechanisms.
Again, I'll skip to the point,
and Betty Pace helped us with this study.
And there's this dual pathway of AP1 as a transcription factor,
regulated by the biochemistry, that turns on cathepsin K.
And then, through NF-kappa B,
is the shear stress-modulated pathway.
That both need to be present for the enzyme to be turned up.
So, that was all our work with human cells and in vitro work.
So, then we began to use the Townes sickle cell mouse model
to see if we could see some of these same things
in a preclinical trial model, or preclinical model.
And because we were worried
or wondering about these young children
showing up with these artery damages and strokes,
we first sacrificed --
or euthanized --our mice at one month,
isolated their common carotid arteries,
and did a van Gieson staining looking for elastin.
And just at one month -- no other interventions,
just having sickle cell --
we see these elastin breaks in this degraded elastic lamina
in the artery wall one month of age.
And we could quantify the elastin breaks over time.
Then we did immunohistochemical staining for cathepsin K.
And you could see here there's a fact where the SS mice
are expressing a lot of cathepsin K in the artery wall.
Remind you -- an elastase and a collagenase --
right around all that elastin and collagen there.
And so, Betty Pace is also my student's committee
and she knew about the JNK data.
She was like, "Why don't you inject that in the mice?"
I was like, "That'll never work clinically.
Well, we need something -- just go for it and let's see."
So, Phil wonderfully did daily injections of this JNK inhibitor
in our sickle mice from one month to three months.
No, they did not like it.
But we looked for what are the systemic
what are the effects with the systemic inhibition.
And what we saw was, like,
so now from one to three months of age.
So, by three months, we see that expansive outward
remodeling of the carotid artery in the SS mice.
But the JNK inhibition actually stops that morphology changes.
We looked at cathepsin K, again.
Three months -- cathepsin K is up in the artery wall,
but with the JNK inhibition,
we had severely reduced the signal of that enzyme.
We also then looked at mechanical testing
of these arteries.
Would that play? If I punch it -- so we can play.
But as you can tie cannulate the carotid artery,
increase the pressure through -- the video's not playing,
but you'll see the artery wall expand, right?
So, the stiffer the artery is,
the less expansion you see as you increase pressure.
So, we look at these pressure-diameter curves,
and our hypothesis was that
if the elastin is being degraded --
that confers compliance to the artery wall --
then we would see stiffer vessels
in the sickle cell arteries.
But we actually didn't at one month, nor at three months.
It's actually the sickle arteries were more compliant.
So, what does that tell us?
Well, there's more than just elastin
that is conferring mechanical properties to the artery wall.
So, my wonderful research scientist, Hannah Song,
actually then looked at collagen in the artery wall.
So, we went and did Mason trichrome staining.
And what's really shocking is that top image on the right
with Mason trichrome, where you see blue,
is where collagen is present.
And then that SS artery -- these are now at five months.
So, we did the drug treatment
from three months to five months.
Five months, that elastin is gone in that artery wall,
and you can see it's all red.
Plus, you can see the expansive, and it almost looks aneurysmal.
But again, with our JNK inhibitor treatment,
we can actually retain some of the collagen
in that artery wall. Collagen, remember,
confers tensile strength to the artery wall,
which now we're understanding
why we're seeing more blown-out compliant arteries
in the SS mice.
So, as we look at these things happening over time,
at the early stages,
we're seeing early elastic lamina damage,
which we might put there.
And then at three months, we're seeing greater elastin breaks.
And by five months, we're seeing this loss of collagen.
So, we're seeing these enzymes that can degrade
both of these structures be increased over time.
How we relate that to the human condition is that, again,
it's known that at the earlier ages,
children are at greater risk of a thrombotic
or an infarctive stroke, versus at the later ages,
people have a greater risk of hemorrhagic stroke.
So, if you're reducing the mechanical integrity
of that artery wall,
greater susceptibility to rupture.
Lots of variability, as has been mentioned.
And we think about that -- I do -- for other work.
I do personalized medicine and other things.
And even in our mouse models,
there is great variability of the disease severity, right?
In the humans, you see 11 percent get a major stroke.
So, it's still variability with all.
So, my wonderful scientist, I found a protocol
for label-free magnetic resonance angiograms,
so we could actually track the same mouse
over time versus these endpoint analyses
and averaging it all together.
So, we began to do MRA -- a wonderful grad student
who started about three years ago now --
where we could do a label-free MRA
on these sickled mice and track the same mice as they grew one,
three, five, seven months.
Reconstruct the arteries, again, in computer,
and then make morphometric measurements
to watch it change in a mouse over time,
with or without sickle cell. And what we did see at first
was what we found histologically was supported,
in that in the SS mice,
you do see expansion of these lumens as these mice grow.
There actually is a bit of a sex-specific difference.
The males steadily get worse,
whereas the females kind of narrow off
by three to between three and five months.
Which again, in the humans,
the males have a lower life expectancy than women.
So, we're seeing some of that reflected.
But what really was nice with the MRA allows us to see
is you actually can see in one artery in a mouse
a development of a stenosis between one of our time points.
And why that's interesting is when we look at measuring
the way we get this measurement,
we measure the artery diameter along the full length.
You can actually see it looks like the arteries
shrink over time, but it's not that.
There are now stenosed regions in the middle that,
as you average it out,
make it seem like the vessel's getting smaller.
So, we're looking at better ways to analyze this morphometry.
But again, taking into account the variability,
this now allows us to now look at how are
these things different, though they all have SS.
And so, one of my talented post-bacs at NIH
has been analyzing these.
And we see that in some of the SS mice,
this one here, there's variability.
One of these mice at three months, no stenosis.
And at five months, you can see a stenosis
in the carotid artery.
Versus a separate SS mouse at three months,
had a significant stenosis that persisted at five months
and didn't resolve itself.
Versus AA, we never were able to visualize that.
So, we now put up a new workflow
because, again, I told you earlier,
with a stenosis, you get changes in the blood flow patterns
and regions of flow circulation.
We want to now model that with computational fluid dynamics.
So, this is our workflow there after we get the MR,
a series of softwares.
And we can then -- it's going to play by itself,
it's going to skip, but we're good --
and so, we can recreate these for multiple vessels.
And again, what's unique about sickle cell,
we are looking at disturbed flow
in the straight parts of the carotid artery.
We know we're going to see it in the bifurcations,
but it's that straight part that makes sickle cell unique.
And we are able to localize these regions.
Also, I guess I'll play with -- push it -- I'll push it.
I'm scared to push it because I don't want to go, okay.
Where we can also then calculate what are these areas
of low or oscillatory shear stress,
which occurs as these sites of disturbed flow.
And those are then subject
to the upregulation of these proteases
that will remodel the artery wall.
Right, now that's a beautiful image, as you can see.
I'm not this fancy; they made these wonderful things for me.
But what we found in the SS arteries
is that you actually get this variability around --
or this asymmetry around --
even the straighter parts of the common carotid artery.
And we can then --
you can see the greater asymmetry in SS versus AA.
But we also then now have able to match the MRA.
Then when we sacrifice or euthanize the mouse,
we can then look at the histology
on that exact artery of which we did the CFD.
Because, again, to reduce that subject-to-subject variability
by looking at one subject at a time.
And what we see with the asymmetry, you see --
again, I'm sorry about the laser pointer --
but you see at this part
where you have these straighter parts of the elastic lamina,
that's where you have the elastic lamina breaks
and the nicks that straighten it out.
But where you get that nice wavy part,
that's the intact elastic lamina.
And where we get the regions of disturbed flow
and low oscillatory shear,
which is asymmetric to the artery wall,
that's where we're seeing these uniform breaks
in the elastic lamina.
And then we then wanted to match it up with genes
that we know are shear-stress regulated
to confirm where we get the elastin breaks.
We are now seeing the genes that have been established
to be regulated by flow. We haven't done ENOS yet.
But cystatin C was one that I also did during my PhD.
It's elevated under unidirectional healthy flow.
It is actually the inhibitor of the cysteine
cathepsins that the cells make.
So, the inhibitor is highly present
in the curlicue part of the elastic lamina
and is actually not as much expressed
in the straighter nicked part.
And so, again, just validating that where
we're seeing shear-stress gene protein expression
is where we're seeing the low and oscillatory wall
shear stress,
which is where we're seeing the CFD that indicated it.
So, just to close all that up,
these anatomical changes we've been seeing in the carotid
and cerebral arteries do change the fluid dynamics
and induce flow-mediated arterial remodeling.
And if we can look at some of these other disturbed
flow-mediated proteins
that we know can remodel the artery wall,
it opens up new therapeutic possibilities
to protect these arteries for children with sickle cell.
Again, the mechanics of blood flow integrates
with the inflammatory and biochemical cues,
which make it a wonderful challenging problem,
along with all the other challenges
that we struggle with with sickle cell.
And again, this patient-to-patient variability
is also reflected in artery damage.
And we'd love to partner with clinicians
with longitudinal MRA data
as we can take what we were doing in mice --
can we transfer this to humans --
and you all would have the outcomes that would be,
again, a better validation of what we might be seeing.
Thank you for your time,
the initiative I allowed [laughs].
[applause]
I want to thank my funding sources
from before I moved to NIH.
Now, my funding is all from NIBIB.
I've got a great group that has now been grown --
sorry, grown -- there at the NIH.
And I love this picture at the bottom.
That's us at the Sickle Cell Walk Run
for the Sickle Cell Foundation of Georgia two years ago.
And appreciate them for all their help.
All right, thank you.
[applause]
Female Speaker: Thank you, Manu, I'm sorry I have to compete.
I don't know what's going on.
Manu Platt: That's all right,
as long as I don't hear it on this video.
Female Speaker: We do have five minutes for questions, Scott.
Scott Pestelak: Scott Pestelak [phonetic sp],
UPenn. Really great talk.
And I too am shocked at how early patients
with sickle cell disease develop
cerebral vascular malformations and stroke.
It's really impressive from a biological perspective,
and your work was really interesting
from that perspective to be able to figure that out.
Do you think, you know,
the only way we know how to treat patients
who have had stroke with sickle cell disease
is by longitudinal transfusions and exchange transfusion?
Do you think any of the work you're doing
or there's any evidence
that we could be able to use this to understand
how to remodel in some way to reduce long-term risk?
And if we could use that to be able to risk
stratify patients as well?
Manu Platt: That is a really interesting question.
We have thought about, in our mouse models,
that we want to try transfusion protocols
just to see if we can also prevent this arterial
remodeling with the transfusion protocol
that would match up to what's in humans.
And we don't have that answer,
but it's something that we could think about
because we have now shifted -- I don't show this here --
we started looking at transplantation.
And even after BMT, is the artery wall
still protected from further remodeling?
And from that aspect, we have seen that early transplantation
does prevent further artery remodeling.
But if we wait too late, the artery's already damaged,
and there was no protective effect,
which I would imagine would transfer the transfusions.
Scott Pestelak: Yeah, interesting. Thank you.
Manu Platt: All right, thank you.
Female Speaker: Bob has a question.
Male Speaker: Hi, I was just fascinated
by the anatomical changes.
And I'm wondering about monocytes, macrophages,
and their role. Lenzon [phonetic sp]
just recently had an article in Science dealing with dooming
and grooming of hematopoietic stem cells.
I don't know if you saw that article or not,
but it's a mechanism by which senescent hematopoietic
stem cells are removed early on.
And I'm wondering, in sickle cell patients,
there's a lot of red cell removal
because of the sickle cell.
Is there an association between your plaque formation
and red cell removal?
John, for example, has done studies
looking at the pharmacokinetics of red cell clearance
and found that the sickle cell patients
had a very rapid loss of red cells.
And I'm just wondering
if that's the mechanism that's occurring --
is that the macrophages are getting activated
and causing the endothelial damage?
Another question would be,
do you see increases of C-reactive protein
in these patients that develop stroke?
That's an athero question -- the C-reactive protein.
Manu Platt: Another thing, maybe --
we have not yet translated that to sickle cell.
So, not looked at CRP yet.
As far as red cell clearance, we don't address that.
I'm happy to talk after this
about how maybe there's some collaboration
that we can do there.
But one thing we do care about --
we expected, actually, that once we opened up these arteries,
we would see monocytes adhered all along the endothelial layer,
which we have not seen in the way that we perfuse
and process our samples post-euthanization.
So, one way we are trying to look at then
the monocyte interactions in the artery walls,
there's trying some new imaging modalities.
But one thing we are going to do directly --
we now have approval for animal studies
to put in cranial chambers in these mice.
And we've been crossing the Townes mouse
with the GFP monocyte mouse
so that now we can have the two together
and actually then look at monocyte attachment
rolling to look at the amounts of monocytes
and how long they actually stay attached to that artery wall.
But there's some complications --
or there's, let me say, controversy --
about does the shear stress in the arteries
allow the monocytes to stick or adhere?
But I'm like, they do that under an atherosclerotic regime.
Why would they not under these conditions,
even with greater activation of the endothelium?
So, that's where I can try to get back to you
and see what those monocytes do.
Male Speaker: Well, that's really a good way
of approaching it
because you're comparing what you normally see
with what you'll see in the sickle.
Right, thank you.
Female Speaker: Okay, I think we will close this session.
Thank you so much, Manu. Manu Platt: Thank you.
[applause]
Female Speaker: So, I know
it's going to cut into your lunch break,
but we have to try to catch up in the meantime.
So, please come back at 12:45. It's on the program.
Female Speaker: We are about to start,
and Dr. Ramsay is going to take over.
But I just wanted to say, while you're eating,
just to bring your attention
to two tables to the side of hardworking people.
One is a display from the Sickle Cell Unit in Jamaica,
just because you're here in Jamaica.
It's a pity that you couldn't all come
and visit us at our unit,
but we have some resources here that we use at the unit.
So, if you have any time after lunch, or during a break time,
or at the end of the day, please do go and take a look.
And I just want to say one more thing,
that the University of the Western Days,
the Sickle Cell Unit, is very grateful
for some really important support
we have gotten from four sponsors
that enabled the Sickle Cell Unit
to do our little bit to bring this conference together,
and one is represented here.
Zachary is here from Pemex, and we are really very excitedly
looking at the demonstration of the Gazelle.
And our other sponsors who have supported the unit
for this conference
are Fulcrum Therapeutics, Adios, and Bluebird Bio.
Really grateful for their support, thank you.
So, do go and visit the two setups
whenever you get a minute, thank you.
Zachary Ramsay: Hi, good afternoon, everybody.
I'm Dr. Zachary Ramsay, and I'm happy to be chairing
the third session for this afternoon.
So, the third session will be on the complexities
of the common complications in sickle cell disease.
And our first presentation will be on insomnia
and sleep health by Dr. Monica Hack.
Now, Dr. Hack is an Associate Professor of Neurology
at Harvard Medical School,
and she leads the Sleep and Pain Laboratory
at Beth Israel Deaconess Medical Center in Boston.
So, Dr. Hack.
Monica Hack: Thank you.
Zachary Ramsey: I was telling you that this is
to change the slides.
Okay. And then there's a pointer here if you want.
Monica Hack: Well, thank you very much.
I'm so happy to be here and talk about sleep and insomnia.
And I hope you stay all with me after this, after --
during this nap time, post-lunch nap time.
So, I first would like -- so, I will talk about insomnia
and sleep health. And we'll focus on pain.
So, what I would like to do is touch base on four topics.
What is sleep health? Why is it important?
How do we measure it?
Insomnia -- what is the prevalence of insomnia?
I would like to concentrate on this perpetuating cycle
between insomnia and pain, talk about mechanisms.
What are the systems and mediators
through which poor sleep can exacerbate pain?
Then, how can we treat it?
So, how do we define sleep health?
And sleep health is a multi-dimensional
construct with all dimensions associated with outcomes.
And just to give you some examples:
Sleep health is important for optimal brain function,
metabolic clearance, as well as cognitive function,
adaptive and innate immune system.
So, it's important for infections,
to control infections,
inflammatory homeostasis, metabolism.
Sleep health is associated with the cardiovascular system.
That probably has been most studied.
And, for example, there is arterial regeneration
during sleep that's related to the blood pressure dipping
that we see during sleep.
And also, it's important for somatosensory processing,
in particular pain.
So, when we look at sleep health,
many people think about sleep duration,
and that probably has been most studied.
We do know that sleep that is too little --
as sleep that is too long --
is associated with increased mortality risk.
It's about six to eight hours is the optimal amount
for the majority of adults.
And then another independent aspect is regularity of sleep.
So, this means going to bed,
getting up out of bed at the same time,
plus minus 30 minutes. This is an increasing problem,
irregular sleep patterns in society,
which is an independent risk factor for mortality
and all kinds of other health outcomes.
And then we also have timing of sleep.
So, sleep should fall into the sleep window,
and that's disturbed, for example, in shift workers.
And also, satisfaction with sleep.
So, how is sleep perceived? How is sleep quality perceived?
Do I feel that I'm refreshed in the morning?
Is an important dimension,
and it's, for example, for mental health,
it's more important than the duration of sleep.
And then we have oxygen.
Good breathing is important for sleep health,
and oxygen saturations of lower than 90 percent at night
is the most predictive measure of mortality.
We have efficiency, so this means continuously
sleeping through the sleep period.
So, without having problems falling asleep,
sleep can be maintained without frequent awakenings at night,
which are classical symptoms in insomnia disorder.
And, of course, there's then also alertness
and sleepiness during the day,
which is often one of the very first indicators
that something is wrong with sleep.
So, how do we measure sleep health and sleep?
Of course, there are several questionnaires
--that's probably the easiest way.
There are sleep health screening questionnaires
that cover all these dimensions. In the United States,
the PROMIS Sleep Disturbance Scale is very popular
because it gives you a T-score
that can determine whether a person is below
the population average or above. And then, we have also sleep
disorder-specific questionnaires,
such as the Stop-Bang for Apnea Screening
or the Insomnia Severity Index.
And then, of course, we have sleep diaries.
And with electronic versions,
it's very easy to get a very precise
or more precise idea of these various sleep dimensions.
Sleep diaries are generally filled out every day,
and you can really see what happens
within a longer time period.
And what's getting more and more popular
are wearable sleep monitors.
They can objectively assess the duration of sleep,
regularity, efficiency,
breathing, heart rate variability.
So, they're multi-sensor devices,
they're getting better and better.
And here, you have these ring devices,
such as the Aura Ring or Fitbits.
And there's also -- these are commercial devices --
but there's more and more research on research devices
that can be used for research
and where data can be customized better.
And, of course, then we have, what I wanted to mention
is that really shows the popularity in the United States.
It's about 35 percent who track their sleep
through these devices,
and about two-thirds who change their sleep
based on what they have learned.
And then, of course, we have polysomnography,
sleep apnea tests that can be done not only in facility,
but also at home.
And there are lots of devices now
where you can measure sleep in the at-home environment.
And these are indicated for certain sleep disorders
to quantify breathing-related disorders,
restless leg syndrome, nightmare disorders, and other disorders.
So, as I said, insomnia is highly prevalent,
and it's about 22 percent of individuals
in the United States, Europe, Asia,
Africa meeting diagnostic criteria for insomnia.
So this is diagnostic criteria.
This is not just having one or two insomnia symptoms.
Diagnostic criteria include dissatisfaction with sleep.
And these are associated with difficulties initiating sleep,
maintaining sleep, and early morning awakenings.
And these problems have to cause clinically significant distress
or impairment of function, such as in social, occupational.
And there's also frequency criteria.
So, the sleep problem has to occur
at least three times per week for at least three months
and can, of course, not better explained
by a coexisting disorder or drug abuse.
And, but as I said, these are diagnostic criteria.
22 percent -- there's many, many more people who do not fulfill,
who have insomnia symptoms,
do not fulfill the diagnostic criteria.
So, insomnia is common in a wide range of disorders
involving pain.
It's 72 percent of chronic pain population
that also suffers from insomnia, comorbid with insomnia.
Autoimmune diseases are very common with insomnia.
Post-virus syndromes such as long COVID --
sleep disturbances,
pain are among the top five symptoms in general.
Infectious respiratory illnesses
and also in blood cell conditions,
comorbidity between insomnia and complaints
and sickle cell estimated
between 47 percent and 57 percent.
There's unfortunately not that many studies,
so I found three studies.
So, but what I would like to concentrate on
is really this perpetuating relationship
between insomnia and disease burden.
And insomnia in itself can increase disease burden.
I will here focus on pain.
And, of course, pain increases insomnia.
I mean, pain is a powerful disruptor of sleep.
And the question is that I would like to focus on here is,
how does insomnia contribute to disease
risk and greater symptom burden?
And can we make it better if we treat insomnia?
So, one thing that I would like to go into
is that I think is very important,
is considering sex as a modulator in the association
between insomnia and symptom burden.
What we do see is symptom burden in many diseases
is much more common in females than in males.
They also have more persistent symptoms.
And this also has been reported for sickle cell disease.
And that can have many reasons: chromosomal changes,
cultural changes, social changes.
But also insomnia seems to be a contributing factor.
So, insomnia seems to be strongly associated with symptom
burden, greater symptom burden in females than in males.
That has been shown in various epidemiological studies.
And I would also like to show you
some experimental studies where this has been shown.
So, to better really understand the effects of insomnia on pain,
on inflammation, on cardiovascular system,
we can simulate insomnia in the laboratory setting.
And in the very beginning,
people have used acute sleep deficiency models
of total sleep deprivation, one night of sleep restriction.
And I'm not going into that, but more recently,
people have used more chronic sleep deficiency models.
And they have a higher ecological validity.
So, for example, so the blue basically is the sleep.
So, we can mimic patterns of sleeping very little
during those workdays
and then catching up on sleep on the weekend,
which I think many people do.
And see what kind of consequences does this have
for pain, for inflammation, for cardiovascular regulation.
And then also --
and I will focus a little bit more on that model
is we can repeatedly disturb sleep
with intermittent recovery periods.
So, and the nice thing with these models
is we can really understand the symptom responses:
pain, fatigue, biological responses.
Can they actually adapt to those?
Can we adapt to sleep disruption?
And also, do we recover from these sleep disruption models?
So, this is basically a model
I would like to show you some more data from.
This is a 19-day model.
So, people stay in the clinical research center for 19 days.
And what you can see,
it starts with three nights of undisturbed sleep.
So, people can sleep eight hours per night,
and they have then these three days of sleep disruption.
So, they have 40 minutes of sleep,
nurse comes in, 20 minutes of awake,
40 minutes of sleep, 20 minutes awake.
And then they have a recovery night,
and then they do this again, recovery night, do this again.
And then they come back and have every day,
every night, eight hours of sleep undisturbed sleep.
So, this is the environment.
So, they stay in the clinical research center.
They have a nice room with view of Boston.
And outside, you can see the control.
So, we do PSG, blood draws can be done through the wall.
And these environments are very controlled.
So, food is controlled, fluid, lights is controlled.
But we try to keep it still not like isolating people,
so they can have visitors, access to email and phone,
and so on.
So, this is basically a hypnogram of such a night.
So, if you haven't seen this --
so basically, what you see here is there is wake,
and then you see the different sleep stages.
"R" stands for REM, and then you have N1, N2, N3.
N3 is the deep sleep.
So, they stay awake, they stay in bed,
they stay awake for an hour. And then they go into sleep,
and then they have a wake period.
20 minutes awake, they go into sleep again, 20 minutes awake.
And this is very common in the general population,
and especially in chronic pain population.
This discontinuity of sleep,
I mean, what we cannot model is the problem,
but we can model the sleep phenotype.
So, I just want to first show you how people respond
with respect to fatigue.
So, every four hours, we ask on computerized rating scales
how they feel with respect to fatigue,
pain, sleepiness, and so on.
And here you can see the fatigue responses.
The red is the disturbed,
and, of course, people are more fatigued.
And then the shaded areas, that are the recovery periods.
So, you can see, after one night of recovery sleep,
fatigue goes down.
What I would like to show you is that females and males,
they have the same level of fatigue.
But what you can see in females, they just don't recover.
So, even after three nights of sleeping eight hours,
they're still fatigued. And I think that's important,
because fatigue is such a big problem
in many chronic diseases.
And these diseases are often over-represented in females.
So, when we look at spontaneous pain,
so this is a composite score.
What I would like to show you here
is that the pain during the nighttime
is much higher than during the daytime, right?
And that's something that we can see for all localized
and non-localized pain items.
So, here you can see stomach pain,
headache, back pain, muscular pain,
joint pain, generalized body pain.
And this may mean that pain management
may specifically target nighttime pain.
For example, through timing of energetic medications.
So, we can ask for spontaneous pain,
but we also can look at evoked pain.
So, we use stimuli, pressure pain,
or heat pain here through an algometer.
And what we do see here, this is now sex differences.
So, pressure pain thresholds in general go down,
but here it's only in females.
And then when we look at heat pain,
it's only affected in males.
So, the point that I wanted to make here
is that we always have,
if we want to find better mechanisms,
better therapeutics, more precise therapeutics,
I think we always have to take into account
that there might be sex differential effects.
So, and, of course,
we also see this in insomnia disorder, right?
This is a very young group of people with insomnia disorder
that have not yet developed pain
or are not comorbid with any disorders, not on medications.
And even here you can see there's higher pain frequency,
and there's much lower pain thresholds.
So, there's good evidence that insomnia itself
can increase symptom burden,
and it might be stronger in females than in males.
But what are the mechanisms and potential mechanistic pathways?
So, what I would like to show you is the various systems
and mediators that have analgesic properties
on the very left, hybrid properties --
analgesic as well as hyperalgesic --
as well as hyperalgesic properties.
And the point that I want to make here
is that all these systems are affected by sleep.
For example, opioid system.
Opioid system in a sleep-disturbed person,
the responsivity to exogenous opioids is reduced,
meaning they need more opioid consumption.
Melatonin has analgesic properties,
and it also has sleep-promoting properties.
So, there's lots of studies -- not lots,
but there are more and more studies out there --
really looking at whether through melatonin
we really can target this association
between insomnia and pain.
And, of course, the immune system
probably has been best studied.
There's a clear upregulation of pro-inflammatory mediators,
as well as prostaglandins.
And I would like to talk a little bit more
about two promising mechanisms,
and that's central pain modulation and inflammation.
So, central pain modulation has been --
these two mechanisms have been probably best studied
in the context of insomnia and sleep disturbances.
So, central pain modulation, basically,
there is a network of cortical structures
that project to the dorsal horn
where a sensation can be inhibited or facilitated
before it reaches consciousness.
So, it has been shown that these descending pathways
are dysregulated in many chronic pain conditions.
And they are involved in the transition
from acute to chronic pain.
And when we look at the effects of sleep disturbances,
there are less effective pain inhibitory circuits,
in particular in women,
and more active pain facilitatory circuits.
And then we also have inflammation.
As I just mentioned, there's various inflammatory markers
that are released in sleep-disturbed people
that are able to sensitize peripheral nociceptors,
as well as central pain transmission neurons,
and then lead to an exaggerated pain response.
And these include, basically, all kinds of pro-
and counter-inflammatory pathways:
NF-kappa B pathway with a production of cytokines,
cyclooxygenase pathway,
hypothalamus-pituitary-adrenal axis
with a factor hormone cortisol,
and also the so-called -- that's pretty new --
the so-called specialized pro-resolving mediators.
That there's very little human research out there.
They are derived from omega-3 fatty acids.
But they have shown in pre-clinical animal models
that they have high energetic efficacy
in various clinical models.
But we are not there yet in humans.
So, okay, and, of course,
there is, like, there are interactions
of these inflammatory and neuronal pain pathways.
But they also determine what kind of pain we then have:
nociplastic type of pain that's more regulated to --
that's more associated with the processing of pain --
or more nociceptive type of pain.
That then also can, if we know this,
that can inform treatment decisions.
So, how do we break the perpetuating cycle
between sleep and disturbances and pain?
Of course, we can target mechanisms such as inflammation.
There's very little data out there
that really looked at insomnia and pain in parallel.
We can target pain through NSAIDs,
corticosteroids, opioids.
But here, we'd like to more focus on, can we target sleep?
And if we target sleep, can we improve pain?
There are various pharmacological approaches --
non-benzodiazepine approaches that,
in general, have a better safety profile,
less dependency than compared to benzodiazepines.
There are orexin receptor antagonists, melatonin,
as I said, has both analgesic and sleep-promoting properties.
And also cannabinoids.
And I think we hear later more on cannabinoids.
What I would like to focus on a little bit
is non-pharmacological approaches,
cognitive behavioral therapy for insomnia,
or hybrid therapies that target both insomnia and pain.
And then what's also coming more and more
is integrative mind-body therapies,
such as mindful movement, yoga, tai chi,
qigong, that just have --
they have a lot of these non-behavioral therapies
have a lot of acceptance because they have a very,
you know, they just come with less side effects.
So, does treatment of insomnia with CBT-I improve chronic pain?
Cognitive behavioral therapy for insomnia
is the first line of treatment for insomnia,
and it outperforms pharmacotherapy
because it has longer-lasting effects.
And then there are different --
so, the traditional delivery method is face-to-face,
but there's now many digital apps, such as Sleepio,
ShutEye, or CBT-I Coach,
that have been validated in clinical trials.
And it has various components that I will not go into it.
It's sleep hygiene -- so, avoiding screen time
before bedtime, stimulus control.
There are sleep restriction components
to increase sleep efficiency at night.
They have relaxation components, and also cognitive components,
such as restructuring of undesired thinking patterns.
So, this was the title of an editorial,
is Cognitive Behavioral Therapy
for Insomnia the New Best Painkiller?
And that was an editorial on a study on a meta-analysis
of 14 high-quality randomized clinical trials
in people comorbid with insomnia and chronic pain:
fibromyalgia, osteoarthritis, shoulder pain,
back pain, and other pains.
And they showed that there is a significantly higher probability
of having better sleep at post-treatment
as well as at follow-up.
When we then look at pain, when we compare baseline pain
with post-treatment, 58 percent probability
of having less pain at post-treatment.
But then, when we look at follow-up,
this is not significant anymore.
So, this also shows that we probably need --
for targeting chronic pain comorbid with insomnia --
we need combination therapies. And I also would like --
sleep intervention has been frequently studied
in the context of surgery, because post-operative pain
is such a major healthcare challenge
that remains under-managed. And also, what we can see here,
they actually used pharmacological interventions,
and we see that with the improvement in sleep,
we have a reduction in pain
and also decrease in the amount of analgesic drugs
in these surgery patients.
So, what are the clinical implications?
You might think, why did I not present anything
on interventions in sickle cell disease?
There are no studies out there yet.
Unless -- I don't -- I think there is, like,
there's great awareness of the importance,
but so far -- and I hope this will come soon.
So, what are the clinical implications
in this perpetuating relationship
between insomnia and pain?
And one, of course, is monitoring sleep health through,
you know, it's pretty easy -- to wearable devices
that often give feedback on sleep-wake patterns.
And it's important to promote sleep health
early on through behavioral and/ or pharmacological interventions
or combinations of interventions to protect from pain development
and better manage pain.
And that's not also true for pain.
That is also true for many other health outcomes.
And I think what I also would like to say
is that sleep problems have often treatment priority
in individuals with chronic pain.
So, they are much more determine quality of life
than other aspects of pain.
Of course, healthcare providers may screen for insomnia symptoms
in pain clinics on a regular basis.
We have to be careful with sleep-disturbing medications
and analgesics such as opioids.
They can, by itself, form like a vicious cycle.
And I didn't talk about this -- considering timing of medication
to minimize sleep-disturbing effects,
considering sex to really have more precise
intervention methods, better understand mechanisms
that link insomnia and chronic pain --
that would then be the basis to really develop more novel
and more targeted
and personalized therapeutic targets.
So, thank you very much for listening.
I'm still in time.
[applause]
We have questions now.
Male Speaker: Yes.
So, thank you very much for that great talk.
Welcoming questions. Five minutes of questions.
Male Speaker: So, thank you. That was really interesting.
I'm wondering if you could unpack the recommendation
about screen time and sleep hygiene.
So, it's a recommendation that we make all the time,
but a lot of patients will tell you,
you know, I like to read before I sleep.
So, I read on my screen.
I don't recall us recommending decreasing book time
before there were screens.
So, is there a way to unpack that?
Is there a healthy way to use your screen to help you sleep?
Because many patients do use their screen to help them sleep.
Monica Hack: So, there is, I think, one option is to have,
I think many phones and many screens have filters, right?
So, you filter out blue light and other wavelengths
that will help you to not,
to keep circadian rhythms in place.
So, that would be one option. But I completely agree.
It's not -- it's not -- because this is also
for many people a relaxation method, right?
So, they, before they go to bed, they just read for 30 minutes
or watch a relaxing show or something.
But that's probably the best option that's out now.
Male Speaker: [inaudible]
Monica Hack: And. Yeah.
Yes. That has been shown. If you really reduce, use those.
That has been shown to, you know,
to less interfere with circadian rhythmicity.
Female Speaker: Hi. Thank you so much for your talk.
I was wondering if you could give us
a little more of the background of the participants of the study
that you mentioned that were on
for 19 days with disrupted sleep.
We're very curious in learning, at least for me,
if any of these patients had sickle cell disease.
We saw that they were distributed female and male,
but a little bit more of the background
and how they were enrolled.
And the second aspect of my question,
since we don't have studies in sickle cell related to sleep,
do you have any --
there's so many things that we can study --
but is there anything that, in particular,
interests you that we could start looking at?
Monica Hack: So, with respect to the study that I showed
on these experimental in-laboratory designs,
these are healthy individuals. They're healthy individuals.
And the reason for that is to really understand --
you start with a healthy individual
to see what sleep disturbances in itself
are doing without the confounding effects.
You know, that's now -- that's getting complicated.
If you have real patient, you have medications,
you have so many other comorbidities,
you need a huge sample size to really figure out
what's the contribution of sleep.
So that's the reason.
But of course, there is very limited generalizability, right?
I mean, you know, okay, sleep is doing this in this context,
but now it really needs to go out.
What do we actually see in much more complex people
who are sick, right? What can we do?
So, with respect to sickle cell,
I think I'm not aware that there are currently any studies
on any kind of CBTI treatment.
I think what would be important to really --
and I think there's also very limited literature I found
on what kind of sleep disorders are actually prevalent.
I mean, everybody, there is a lot
on sleep-disordered breathing; there's much known there.
But on insomnia, what kind of insomnia there is,
how many are actually affected, who fulfill insomnia disorder --
I think there needs to be more base research out there
before we can really then target and design therapies
that might be not just CBTI, that.
I think there's a lot of combination therapies out now
with CBTI plus light, plus pharmacotherapy,
plus melatonin that can be very effective,
but I think we need to know what's actually there.
And I think there are still --
I hope this research will come, and maybe I can help with that.
Monica Hack: Thank you.
[applause]
Male Speaker: All right, thank you again
for that great talk.
So, our second presentation is on Pain,
Many Mechanisms, and Mysteries, and it is by Dr. Cheryl Stucky.
So, Dr. Stucky is the director of the Pain Division
of the Neuroscience Research Center
at the Medical College of Wisconsin.
Cheryl Stucky: All right, thank you so much
for that very kind introduction. I really appreciate it.
Yeah, and you even said my last name right.
Stucky rhymes with cookie in my case. Good job.
Yeah, so it's a real pleasure to be here.
I'm from beautiful Milwaukee.
It's not quite as warm as here, but it's lovely.
And I'm here to talk about pain in sickle cell disease.
So this, I feel like, is a really cool --
does this work, this pointer?
There we go. It won't work. Sorry, does it work?
Male Speaker: So this is to change the slides,
but you can use the pointer.
Cheryl Stucky: This is to change the slides?
Oh.
Male Speaker: The red button goes backwards.
Cheryl Stucky: Oh, very good. Okay, great.
All right, so this is a painting.
Have you seen this before? Yes. Who is it by?
It's Hertz Nazaire. It's a self-portrait of himself
when he is in a acute pain episode.
And it is called 10 Redefined,
because on a scale of zero to 10,
with 10 being the worst pain that you ever feel,
it's worse than that. And you can see.
Unfortunately, Dr. -- or Mr. --
Nazaire passed away about three years ago
from complications of sickle cell disease
at the age of 48.
Okay, there we go.
So, as you know, sickled red blood cells are --
when they sickle, they cause vaso-occlusion.
That occurs when the hemoglobin molecules form polymers
when they're deoxygenated.
And sickle hemoglobin, this causes vaso-occlusion,
repeat tissue ischemia, and reperfusion injury.
This also leads to a shortened red blood cell lifespan,
shortened survival.
They lyse their contents into the blood,
dumping heme and other kinds of things into the bloodstream.
And there's also chronic microvascular inflammation.
And a huge hallmark, let us be clear,
is the acute and the chronic pain.
It is horrible, and it plagues patients for their entire lives.
So, this is a painting by artist Ellen Weinstein.
It's The Sickle Boy.
And this is how one patient describes how he feels,
with the snakes coming out of his body all over.
And when you ask a patient,
"How do you describe your sickle pain?"
What do they say?
"I feel like knives are stabbing me from the inside.
I feel a radiating deep pain from inside.
I feel like my bones are breaking.
I feel like there's broken glass flowing through my veins.
And I feel like a heart attack
is happening through my entire body."
So, with sickle pain, we have acute episodic pain
that is triggered by cold, stress, exercise, illness.
So what can these kids do? What can these people do?
Not a lot of normal things that other people do.
Can't go swimming. Can't stand in the cold.
You can't really exercise.
You can't do what the other kids do.
Many of them also have chronic pain.
In fact, about 40 to 50 percent
of these patients ultimately suffer from chronic pain,
perhaps after these repeated vaso-occlusive events
that prime the nervous and other systems to have chronic pain.
Then there's acute pain on top of the chronic pain.
Now we know -- and we'll hear more
about this next from Dr. Darberry [phonetic sp]
-- opioids are still the first line of treatments
for especially the acute pain because they do work.
But we all know they have a host of unpleasant side effects.
They cause dependence.
And for many patients, unfortunately,
because opioids are the only thing
maybe that works in their case for acute pain,
when they go to the emergency room in the hospital,
they're thought of sometimes as -- or maybe often --
as drug seekers. That's really unfortunate.
So, we've been studying sickle cell pain
from the mouse perspective.
And I'm so delighted that Manu has opened us up
with describing what these mouse models are like.
We use both the Townes model that he described
and also the Berkeley mouse model.
They're a little different in their genetics.
I won't get into that.
But we've been using the Townes model
because we think it's a little better
for actually recapitulating what actually happens in a patient.
So, how do these mice recapitulate what happens
in a patient? Do they?
So, the patients have sickled red cells.
The mice do as well. Patients have vaso-occlusion.
So do the mice.
Patients have anemia, as do the mice.
Patients have severe organ damage.
So do the mice.
But do they have chronic touch-evoked pain?
Do they have cold pain?
Do they have vaso-occlusive pain?
Do they have spontaneous pain?
So, these are all questions that my lab and others,
like Kalpana Gupta's lab, have been asking.
So, we know that about 40 percent of patients
with sickle cell disease
experience chronic touch-evoked pain.
Touch-evoked pain all over their body.
So, we started by asking, well,
do the mice experience touch-evoked pain?
Here, what we did is we put the mice
on a platform where we could --
and a plexiglass box over them --
and we can poke the bottom of the animal's paw
with a von Frey filament.
Those of you that are MDs or serve
in a neurology clinic know what a von Frey filament is.
It's used for touching or determining thresholds for pain.
And when you do this, in this case,
and you test the animal's bottom of the paw,
each filament bends at a certain force.
And you can see that over here -- paw withdrawal thresholds.
So, the lower the threshold, the more sensitive they are.
The higher the threshold, the less sensitive they are.
So, compared to controls, sickle animals are chronically
very, very sensitive to mechanical stimuli.
We see the same thing if we poke the bottom animal,
the bottom of the animal's paw with a needle --
a frankly noxious needle poke. So, in this case,
the animals have more responses to the needle stick.
The sickle animals chronically do.
And if we just swipe the bottom of the animal's paw
with a light paintbrush,
the animals with sickle cell disease have more responses.
They just don't like being touched.
And so, mice have chronic mechanical allodynia
or hypersensitivity to light touch.
But as our last speaker, Monica, brought up,
what about spontaneous pain?
And that's really important in the pain field now.
Everybody's interested in what do these pain phenotypes
look like when you're looking at spontaneous pain
because the patients have pain when they're just sitting there,
pain when they're moving around. They don't have to be touched.
So, we have begun looking at -- sorry,
I'm getting my pointers confused --
we have begun looking, using AI, Artificial intelligence,
to look at pain behaviors by looking at the animal's face.
This is called facial grimace.
And so, what you can see in an animal that is in pain --
and this is verified by veterinarians --
you will see that they pull their eyes back,
they pull their ears back,
they push their whiskers forward,
they have a nose bulge.
And these are all elements and evidence
that the animal is in pain. So, we did this grimace test
with this artificial intelligence,
and what we see is, indeed, the sickle animals,
shown in red, have higher grimace scores
chronically than the age-matched control littermates.
And we're doing a variety
of other spontaneous pain tests now,
but this suggests that sickle mice also exhibit
this chronic, ongoing-like pain behavior.
Now, where is that pain coming from?
Well, we know that pain receptors, called nociceptors,
in the body that are all over the body
are what respond to painful stimuli.
So, my lab asked a question:
well, are these pain receptors --
are they actually sensitized?
Do they actually fire more impulses
or more action potentials in response to a touch stimulus?
And so, we took our mice -- sickle animals,
sickle and control animals --
and what we did is we took this hindpaw skin and the nerve,
the saphenous nerve that innervates that skin,
took it out of the animal, kept it happy in a tissue bath,
and then recorded from single primary
afferent sensory neuron nociceptors.
And this right here, as you can see,
the action potentials that you get in a single pain receptor
when you stimulate it with mechanical stimuli.
And so, what you can see
is both the C-fiber unmyelinated nociceptors
and also the myelinated nociceptors respond more,
with higher frequency of action potential firing,
than the control animals --
the age-matched littermate controls.
So, the nociceptors all over the body are sensitized,
and they're sending more input into the body.
Now, question we asked,
can we reverse this pain with TRPV1 inhibition?
Okay, so who knows? Tell me about what this is.
Who knows what these are?
How many of you like hot, spicy food?
A lot of you? Okay, cool. Yeah, hot, spicy food.
Does anybody know what the chemical in hot, spicy food is?
Capsaicin, you guys know.
Okay, do you know why the capsaicin was made famous
in the last three years? Think Nobel. Nobel, Nobel Prize.
Yeah, Nobel Prize was won by this gentleman, David Julius,
for discovery of the capsaicin receptor, the TRPV1 --
transient receptor potential vanilloid 1 -- receptor.
He won the Nobel Prize in 2021. So, this is a really big deal.
It's hot stuff. So, it is hot stuff.
So, just trying to wake you up, right?
So, hot stuff, locally, through patches, does alleviate pain.
So, you've known for many years that you can use capsaicin
as a patch, as a cream, and so forth.
You can inject it into the knee. It causes -- it alleviates pain.
And we can talk about the mechanisms for that later.
Okay, but the point is that if you try to inject capsaicin
inhibitors systemically, systemically, it's a problem.
Many, many people have tried to.
Drug companies have tried to do this.
What? So, these local analgesics are effective,
but systemically, the inhibitors induce hyperthermia --
so, fevers in patients and in rodents.
We can talk about mechanisms later.
And they also dampen the individual's protective pain.
So, if they put their hand on a hot stove,
they won't feel it,
and that's bad because they get burnt.
Okay, so the question is, can we target upstream of TRPV1?
Can we target mechanisms that are involved
in sickle cell disease upstream to TRPV1?
Here's where we started bridging the translational gap.
Because there are basic science investigators like me,
there are clinical investigators in this room,
and then there are these wonderful clinical individuals
who are treating the patients.
And I feel like a lot of times, we're on our own islands,
and then in the sea are all the patients
trying to find help somehow.
And so, what I think we need to do a lot more of
is bridging the gap together -- bridging the islands together --
and I hope that meetings like this will help us do that.
So, what we have done in my lab
is partner up with a fantastic sickle cell clinician.
She's named Amanda Brandow. She's fabulous.
And this is my lab, and we are really trying very hard
to make this bridge of translational research.
How are we doing that? How's our first endeavor?
So, let's let patients guide our research.
So, Amanda treats kids and adults with sickle cell disease,
and she said, let's let the patients guide our research.
Well, how will we do that?
Multiple Speaker: [inaudible]
Cheryl Stucky: Oh, yeah, sorry. I keep getting confused.
There we go.
So, what Amanda did is she took plasma from patients
with sickle cell disease during an acute pain episode
and compared it to healthy baseline plasma,
and then she did a gene-associated array
and looked at pain-relevant molecules.
And these are the hot things that came up.
This is not published yet,
but she's allowing me to present it.
The number one top molecule was tumor necrosis factor,
and we heard about that from Manu already.
So, tumor necrosis factor is a hot one.
Here are a couple of others: CCL2, CCR5, and so forth.
And so, we know from past literature
that tumor necrosis factor sensitizes TRPV1 channels.
So, maybe TNF is upstream to TRPV1,
and a really interesting thing
is that TNF-alpha inhibitors are already FDA-approved,
like infliximab and some other things like etanercept.
So, we started testing infliximab,
and this is now back to our mouse model.
So we asked, does TNF inhibition affect
mechanical hypersensitivity after a induction
of an acute vaso-occlusive event?
So how we did this is the protocol is on the left,
we infused the animals with infliximab for several days
and then induced an acute vaso-occlusive event
with putting the animals in a hypoxia chamber.
And then we tested their mechanical thresholds.
And so what you see on the left here --
again, I want to remind you that the lower end is actually --
the lower end is going to be more sensitive down here,
and then the higher end is less sensitive.
And so our wild type animals
that do not have sickle cell disease are in blue,
and the animals with sickle cell disease are in pink.
And the animals that are treated with infliximab are in solids.
So before any treatments, just with infliximab itself,
you can see our baseline.
These sickle animals are quite sensitive.
Then with the gray stripe there,
that's where we induced the vaso-occlusive event.
So induced a vaso-occlusive event.
And the sickle animals that were treated with vehicle
are in the open circles.
They not only remain really, really sensitive,
but they get even worse, even more sensitive as --
after this vasal occlusive event.
And they continue to be very, very sensitive.
On the other hand,
the animals that were treated with infliximab,
those in the dark pink circles, the filled circles,
they get a complete reversal of this hypersensitivity,
which is pretty interesting.
Then we redid the vasal occlusive event again
with that gray stripe.
The animals with sickle cell vehicle,
they get even worse down there at the bottom.
Those open circles,
they're going to be even more and more sensitive,
whereas the animals treated with infliximab have, again,
a reversal of the pain hypersensitivity,
touch hypersensitivity.
And again, that happened again a third time.
So then, we asked, well, what --
what's happening at a cellular level?
Can we take out the pain receptors,
the nociceptors that are in the dorsal root ganglia
by the spinal cord, put them in a culture and record
from those pain receptor nociceptors?
So here's what we do on the left here,
that's a cell on a cover slip that we're going to target,
and we're going to do whole cell
patch clamp electrophysiology on that cell.
And we simply look at the resting membrane potential.
For example, and on the left,
what you can see is compared to the blue animals,
the control animals, the resting memory potentials
of the sickle animals are depolarized.
So they're more sensitive,
they're more likely to fire an action potential.
And if you take neurons out of animals
that were treated in vivo with infliximab
and do these ex-vivo recordings,
the infliximab reverses this elevation
in resting membrane potential.
Then if you look at spontaneous action potential firing,
the top is -- the top trace is a neuron
just from a sickle animal treated with vehicle.
You see those lines, those are spikes,
those are firings of action potentials in that neuron.
The infliximab treated-animal neuron is below.
There are fewer action potentials,
and if you quantify those,
the number of action potentials on the right side,
compared to the blue control, the sickle animals have way more
spontaneous action potential firing,
and it's brought back down close to baseline
if you take animals
or if you take neurons from the animal sickle animals
or that were treated with infliximab.
So -- and then we asked, okay,
is this happening through TRPV1, the capsaicin receptor?
So in this case we did calcium imaging
where you can basically use a calcium sensitive Fluo-4
and look at neuronal responses with this Fluo-4.
And hopefully what you can see
is this is we're now stimulating those neurons with capsaicin.
And what I think you can see
is that the sickle animals treated with vehicle,
those isolated neurons are more responsive to capsaicin.
And if you take neurons out of the infliximab-treated animals,
that's returned back to normal capsaicin responsiveness.
And the same is true with the amount of calcium influx
when you stimulate them with capsaicin.
So it seems like infliximab reverses the sensitization
of TRPV1 in pain receptors at the cellular level as well.
And again, we did this with whole cell patch clamping
and you can see that the infliximab again
reverses that responsive --
enhanced responsiveness to capsaicin.
And it's similar to if you use a TRPV1
inhibitor on those neurons.
So we think this mechanism is occurring ultimately
through TRPV1,
this TNF sensitization during sickle cell disease.
So we're wondering whether we could possibly think
about repurposing FDA-approved TNF inhibitors
for the acute pain events that tend to be associated
with a vaso-occlusive event in sickle cell patients,
patients with sickle cell disease.
And I'm almost done. We have a lot of mysteries.
We understand so far the tip of the iceberg
for what's causing the pain in sickle cell disease.
And what are the things we don't know?
This is my last slide, but there are many things we don't know.
What are the underlying mechanisms for acute
and chronic pain and sickle cell disease?
We're starting to understand them,
but we still have a long way to go.
How do these account for the individual variability
between patients and their different phenotypes?
What are the risk factors,
but also what are the protective factors
for developing pain over the lifespan from babies to adults?
Well, we have existing therapies and Dr. Darbari
is going to be talking about this next.
So, which of these are the most effective for acute pain
and a chronic pain and then acute on top of chronic pain?
Will early therapy as babies or young children --
will that actually prevent the development of chronic pain?
We still don't know that.
Will bone marrow transplant or gene therapy
reverse chronic pain that's already established?
Will it?
And if it will, at what time and point?
We don't know that.
Can we identify novel ideological targets for acute
and chronic pain? Novel targets?
And that's one thing my love is really, really interested in.
And with that, I just want to thank the members of my lab.
This is the Stucky Lab on the left.
I want to thank my funding sources largely from NIH,
especially NINDS and NHLBI.
And then I really, really want to shout out
and thank my fabulous clinical partner, Amanda Brandow.
And I hope that you all will partner up with a clinician
if you're a basic scientist or a basic scientist
if you're a clinician,
and really try to go bench to bedside and backwards
because I think this is a really strong pathway forward.
And thank you very much for your attention.
Zachary Ramsay: Well, don't go too far.
Yes, thanks very much for that talk.
I actually do a lot of work with quantitative sensory testing.
So this is very exciting
and certainly a lot of the papers that you've --
you know, you're discussing particularly from Dr. Brandow,
you know, they've been very, you know,
useful in guiding a lot of that research.
So I'm going to welcome two short questions.
Just trying to keep up with time.
Male Speaker: Yeah. That was very nice.
And I'll keep it quick.
At what age did you do these pain studies with your mice?
And you hinted at this, at your final conclusions,
and did you see any age-related differences
in their pain responses into the medicine?
Cheryl Stucky: Excellent question.
So we've seen the pain phenotype and chronically by two week,
or sorry, by two months of age,
it gets worse and worse as the animals age to a year.
So it gets worse with age.
We are now conducting studies to look at earlier than two months.
It is kind of difficult because the myelination
doesn't develop after birth for a few weeks
and the myelination is really important
for how they withdraw from the stimulus.
But it's a great question. Thank you. Yes, please.
Swee Thein: I have a quick one.
So I'm really interested, you know,
I'm fascinated by this pain. So you mentioned about TRPV1.
Cheryl Stucky: [affirmative]
Swee Thein: And in fact, I just kind of happened
to look to all this, you know, about the thing.
You know, new -- using a new approach
and I didn't realize that there's so many TRPV1 inhibitors
that are under clinical trials or pain.
Cheryl Stucky: Yes. Yes.
Swee Thein: And so many of them
have been prematurely terminated.
Cheryl Stucky: Yes.
Swee Thein: Presumably for this hyper variant.
Cheryl Stucky: That's exactly why they've been terminated.
Swee Thein: Hyper variant.
Cheryl Stucky: That's exactly why.
Swee Thein: One thing I want to ask you,
why you mentioned the patient variability.
Cheryl Stucky: Yeah.
Swee Thein: And assuming TRPV1 is so important
are they genetic variants?
Cheryl Stucky: Yeah.
There are -- in other populations of humans,
there are -- to my knowledge, it has not been looked at
in patients with sickle cell disease.
I think it's a great question. Great question.
Male Speaker: I have just a quick question.
I -- you know, the TNF-alpha is very interesting
that you see this emerge.
And I'm curious if there's any role
of kind of microvascular thrombosis
or platelet activation in any way,
both in terms of pain and in terms of say,
a risk of thrombosis more broadly too.
Cheryl Stucky: So if you --
you mean with TNF alpha inhibition?
Male Speaker: Yeah.
Cheryl Stucky: That is a fantastic question.
I am not a hematologist. I should start looking at that,
and I welcome your advice on how to do that.
I would love to do that.
We haven't really looked at any other body parts.
It's actually a shame because we sacked the animals.
We only took the one -- the things we knew about.
We could have taken all kinds of things.
Male Speaker: Thanks.
Cheryl Stucky: Yeah. Sure.
Swee Thein: One last question. Cheryl Stucky: Sure.
Swee Thein: So now that we have AI,
can we actually use AI
to actually target just the part that you want,
but you know, avoiding the hyper hyperthermia,
you know, communication?
Cheryl Stucky: Yeah. Hypothermia.
Swee Thein: And hyperalgesia?
Cheryl Stucky: Hyperalgesia. Yeah. Yeah. Fabulous question.
AI is getting more and more developed by the --
by the minute. And so yes, your answer is yes.
We are working on that with other groups
to look at pain behaviors and nuances in pain behaviors.
There's also things like black box recording
where you use AI to look at the gate of the animal
and how much pressure it puts on different parts
and how much it rears up and all.
And you know, the angle of its body.
We're just about to install one of those in our lab.
So the answer is yes, and I think that's going to explode.
Thank you.
Swee Thein: Thank you.
Zachary Ramsay: All right.
And our third presentation is on managing pain beyond opioids.
So looking on cannabinoids, buprenorphine --
I'm sure I pronounced that incorrectly.
And ketamine.
And this is by Dr. Deepika Darbari.
And Dr. Darbari is a pediatric hematologist oncologist
at the Children's National Hospital,
and also the professor of pediatrics
at the George Washington University in Washington D.C.
Deepika Darbari: All right.
All right. Thank you everyone,
and thank you for the organizing committee
for giving me this opportunity.
And as we know, pain is the most common complication
of sickle cell disease. And as we heard earlier,
opioids are pretty much the treatment right now,
but that has many side effects.
So it's important that we learn about some of the other things,
other agents, that are coming up for managing pain
in sickle cell disease.
Just a disclaimer, my first name is spelled
Deepika and Darbari is my last name [laughs].
All right. All right. So these are my disclosures.
So first of all, as I mentioned,
pain is the most common complication.
And as Cheryl mentioned,
it's important to understand their different mechanism.
So when -- whenever we say pain, it could be acute pain,
it could be chronic pain,
or it could be acute episode of pain
on the background of chronic pain.
And the mechanisms are different
and sometimes these mechanism may be present at the same time.
And limitation right now for managing
is our treatments are just opioid centric or NSAIDs.
Those are the one we use.
But all of us you know, who have managed the patient,
we see we can go up on the doses of opioid,
but there's no response.
So patients are treatment refractory
and it's and especially when you have chronic pain.
And we also know that more than 50 percent of the adults
have chronic pain.
So we really have --
don't have many treatment options for those individuals.
So -- and other thing is, if you look at the opioid --
so first of all, opioid is the only choice.
And chronic opioid therapy has become very common,
but that has many side effects.
And the -- you know, some of the mechanism we are --
heard in during the sleep-talk, central sensitization
is one of the very important mechanism for contributing --
of contributing to chronic pain.
So even if you can correct sickle cell disease
with some of the treatments,
you may not be able to reverse chronic pain.
So it's important that opioid can contribute
to central sensitization.
Also poor quality of life, depression.
So all I'm trying to say here
is it's important we look at the other agents.
So these are the three agents I'm going to talk about.
First is cannabinoid, buprenorphine and ketamine.
So first cannabinoid, which has been in news
and has been approved in many of the states in U.S.
So humans have used it for centuries.
Like more than many thousands of years,
this plant has been used.
But in 1960, that was the first time the active compound
or the THC was identified from this plant.
And that led to identification of these receptors.
And these receptors are important
because we'll talk about those. So cannabinoid receptor 1
and cannabinoid receptor 2 were identified.
And at the same time, the whole cannabinoid system
was identified in the body, which is present.
And it's -- the action is through endocannabinoid.
So these endocannabinoids are of two kinds.
And the first one is AEA and 2-AG.
And I want to mention anandamine
because that kind of indicates --
so ananda is the Sanskrit word,
a language, ancient India, that means joy.
So people knew that, you know, cannabis and joy
kind of go hand in hand, [laughs].
But recently -- so people have --
and I'm trying to understand
like what are the therapeutic potential of this medication?
So if you have not seen the plant looks like this,
and there are some of the things which are here,
[laughs], so now next time you see, you know what it is,
[laughs]. So these are the parts.
So one is flowers. So flowers are mostly the one.
Those are used for many forms of edibles or you know,
different ways, but also leaves.
So I learned that leaves are of two kind.
The more potent ones are the small ones,
are the sugar leaves and the fan leaves are important,
but not that important.
And then your stem is also used for many, you know,
like making ropes and stuff like that.
But seeds for the next crop. So plant --
I mean really all the parts of the plants are used.
So the way cannabinoids act
is through two receptors as I mentioned.
So CBR1, cannabinoid receptor 1 and cannabinoid receptor 2.
So the 1 is -- has --
and these receptors are present in our central nervous system.
So important for management of pain because pain
is also felt in our nervous system.
So important thing to know here is that 1 is really --
contributes to a lot of things.
So it's important for pain, anxiety, sleep and appetite.
And that's why we see some of the patients report
that they benefit from using cannabinoid.
But the problem is it also has psychoactive effect.
So that is the downside of cannabinoid.
If we are using or targeting only cannabinoid receptor 1.
Receptor 2 is localized in immune cells.
So the way it works for the pain is by acting on the --
so we also heard in the sleep talk the mechanism
or how the pain is felt.
So it works at different levels. So it acts presynaptically.
So you are -- inhibits the release of neurotransmitter,
changes the uptake and also the activation of DNIC.
DNIC is the -- our natural pathway in our body
that inhibits pain. So it activates pain.
So there are many ways it works in pain
and also in neuroinflammation.
So these are all important thing we know
in sickle cell that's important.
So it can help in sickle cell pain.
But the problem has been the side effect,
the psychoactive side effects.
If we are targeting only cannabinoid receptor 1.
So it's important recently,
CBD or cannabinoid has been used more commonly
and that acts on cannabinoid receptor 2 and not on 1.
And that may have some beneficial effect.
So the -- right now, there are only in sickle cell,
only the mouse model studies that showed that --
as Cheryl showed hyperalgesia. So it can decrease hyperalgesia
and also inflammation important in pain.
So those are the things that have been shown
in sickle cell models so far. How about in humans?
So there are -- some of the studies experienced
from the patients have been reported.
And so really not great benefit at this point,
as you can see here.
So I have listed full study and some --
most of them were like looking at the data
from the medical record or hospitalization
and patient report,
but not really significant difference in patients
who were taking medical marijuana or not taking it.
Only -- there's only one pilot randomized control trial
that looked at the vaporized cannabis
and it did not show any effect.
So that was the only randomized trial
and it actually did not reduce pain or associated symptoms.
So that was the only study. So what can we say?
So American Society of Hematology
came up with the guidelines
on management of pain few years ago,
and their comment on medical cannabis is,
so cannabis at federal level in U.S. is still illegal.
Although states have approved the, you know, medical use
and recreational use, you can use cannabis,
but as a physician you need like special things to prescribe.
So they did not make a comment of like,
would cannabis would work for sickle cell pain or not,
but they did identify it.
So since many of our patients take it,
so it's important that we understand
and we look at does it -- is it beneficial or not?
And especially important not only the efficacy,
but also the side effect
and what are the downsides of using med marijuana or cannabis?
So right now the cannabis status in U.S. is it's schedule 1 drug.
And I have put -- what are --
what is the definition of schedule 1 drug?
So schedule 1 has no currently accepted medical use.
And this is again as the federal level,
so high potential for abuse.
And some of these things you can see here.
But that has not stopped the industry.
So the sales for -- cannabis-related sales
are in billions now, and use has increased.
Other problem is that if you buy those product
from different dispensary, the product may be different.
So there's no consistency and some of that
there will be combination of dangerous substances
that can contribute to side effects.
So it's really -- you know, so some of the --
this research is undergoing,
but we also know many of our patients use this substance.
It's important to educate them about the risk and benefit.
So risk and benefit, the --
these are the side effects, those are listed.
So you can see here pretty much affect many organs.
So paranoia, impaired memory, fatigue,
and some of the things are already present in patients
with sickle cell disease.
One of the biggest concern has been used in adolescent.
So almost like one in three teenagers by age --
by 12th grade they have used marijuana.
And that can lead to multiple problems.
So that has been recently like one of the bigger problem.
And also vaping.
So not only our patients have acute chest syndrome
and involvement of lung,
so it becomes even more important.
And also another thing when teenagers or young adults
are driving while under the influence that can lead to,
you know, harm to them and also people around them.
So it's really important that we think about
and look at the benefits and side effects.
Next, I'm going to move on to buprenorphine.
So buprenorphine is -- it's a -- it's like a opioid,
but it's partial agonist and antagonist.
So it acts on the opioid receptor.
So I have put them up here,
as you can see on the right side.
So the opioid receptor, there are new opioid
receptor kappa and delta. So this buprenorphine acts on --
it's a partial agonist on the new opioid receptor.
So by that virtue it causes pain relief.
So, which is a good thing and, but it doesn't have the --
since it's not full agonist, it doesn't cause those problems
that we can see with the morphine or other opioid.
So like, you know, they don't have dysphoria, sedation.
Potential for addiction is low
because there's not that much of high with buprenorphine.
Depression and other things
are also less common with buprenorphine.
So it has been approved by FDA for management of chronic pain.
It can be used alone.
So buprenorphine alone or buprenorphine with naloxone
has been used.
So this is the picture I have put in,
what's the difference between full opioid agonist
or partial opioid agonist?
So people generally think that --
does that mean it would not help in pain?
But that's not true. It is -- the NLG effects are there,
but the side effects are not there.
So it has become really like --
the use has been coming up for this condition.
Other things are, since the impact,
the respiratory depression and some of the other things
are less, the -- you know, it would not cause --
hopefully it would not cause that much of, you know,
death or overdose death
as the other opioids can do that.
Other important thing is it's hepatically
or it's cleared by liver.
So if individuals have renal impairment,
it can be used for them as well.
So how about -- so buprenorphine has not --
has been used in other painful condition as you can see here,
and also for sickle cell disease.
So how do we do that?
So there are different protocols and you can look up.
So I have put some of the studies up here.
So this can be done,
this transition from opioid or morphine or oxycodone
and things like that can be changed to buprenorphine.
It can be done inpatient outpatient or even at home.
The principle is such that, you know, the solid line
is the opioid agonist.
So it could be long-acting opioid or things like that.
So they -- this person is on high MME or morphine equivalent.
So what you do is this dose comes down
and the buprenorphine dose comes up.
So at some point the patient would be fully
transitioned to buprenorphine
while they are monitored for the withdrawal symptom.
So this -- so this is just the general principle
and there are many path -- many --
there are many protocols that can be used
and the references are in here.
So I'm in pediatrics and what --
how we have been using at our center
is we are gradually coming down on opioid
and slowly going up on buprenorphine
rather than just stopping the drug totally at one time.
So is this beneficial?
So this is some of the data as you can see on the left side.
This is the pre-induction.
As you can see, the patients were admitted or acute care
visit multiple visits. And this is the post-induction.
So you can see the difference was significant.
And so average number from 10
in past six months reduced to three.
So this was like, you know, significant reduction
in acute visit for acute pain.
And patients who continued longer beyond six months,
they continue to have similar effects.
So these were really clear benefit
of buprenorphine therapy.
And listed here is again continuing with the similar,
you know, buprenorphine in sickle cell disease.
So as you can see, the acute care visits,
ED visit, infusion visit,
acute care visit and hospitalization,
they all reduced significantly as you can see pre-induction
and here post-induction.
Other interesting thing that I thought was not
only that acute care visit went down,
but non-acute care visit came up, which is really good
because our patients were not coming to the ER
or coming for pain medication but coming to the clinic more
for their health maintenance visit.
So what -- what was the American Society of Hematology
guidelines set about buprenorphine?
They commented that there is experience with buprenorphine
and other chronic pain condition.
And these studies are research priority,
so it should be studied further,
so can be done safely in our patient population.
So also it's important when we offer a treatment
to our patients,
how would they decide, should I take this medication,
should I give up my opioids or not?
So I thought this was really nice paper.
Looking at this paper interviewed --
the author interviewed individuals
who had sickle cell disease
and transitioned from opioid to buprenorphine
to see what did they think about it.
So they classified this into three themes.
So first of all, patient has to decide like, okay,
I want to do it because as we may say things,
but unless they hear from -- you know,
unless they do their research and decide to do that,
it's not going to happen. Second important thing is here
because if there is some uncertainty,
I'd switch to this medication, would it help in my pain?
And how do our patients get their information?
So obviously personal decision, do their own research,
but also important to hear from other patients
who have sickle cell disease.
So that's important that we educate the whole community,
not just one patient. And also look at the other --
so patients reported the -- their symptoms improved.
So importance of patient reported outcome,
the improved social function, personal function,
and also changes with the pain medication.
They looked at opioid differently and,
but also they identified some of the problems.
And we have faced this in our clinic as well.
Barriers related to like stigma
because the suboxone or buprenorphine is listed
as opioid use disorder drug.
So if you see that medication was prescribed to you,
people may think they may feel stigmatized for that.
And also coverage by insurance. And this has happened to me
because suddenly insurance would decide like,
oh, they need prior authorization
and they had to stop medication, which you cannot do that.
So these are some of the challenges
and some of the benefits that came out from that.
Last but not the least, I'll talk about ketamine.
So ketamine also has been first synthesized in 1960s
and it was tested for -- because it was a fast anesthetic
and a short half-life and relatively safe drug.
So it was used quite a bit in war setting.
So like a battlefield anesthesia and things like that.
But at the same time,
people learned about the side effects.
So delirium, dysphoria, some of these things were identified.
So that kind of, you know, it was not used as much.
In recent year, we have started to think more, you know,
about ketamine because of the -- some of the analgesic property
and things like that, and especially refractory pain.
So it's a dissociative anesthetic.
And the important thing is that it acts on the NMDA receptor.
So NMDA receptor is very important in chronic pain.
And so it's -- that --
this is the population and our clinic also,
we use quite a bit patients who have chronic pain
and develop acute pain on chronic pain.
So that is, it can be used.
Not only it acts on NMDA receptor,
but other receptors or --
and other pathways, it's also effective.
So that's the benefit of that. So it works on opioid pathways
and some of these, as you are listed here.
So ketamine and sickle cell disease.
So keta, if you look up the -- do the search of ketamine
and sickle cell disease, you would see lot of papers.
The problem is everyone has done their own thing,
including our group, looking at like --
you know, so sometimes we all would use it in clinic,
sometimes in ED, sometimes inpatient or ICU.
So really we cannot look at the data together.
And it's -- and also same thing, dosage.
So dose has been variable and root has been a variable.
So intranasal versus IV, et cetera.
Also, then most of the time, how do we decide
when is it time to use? Ketamine is typically left
to the patient physician's discretion.
So first the physician has to decide, now,
my patient has chronic pain, refractory pain,
and they may benefit from that.
We do should not -- we should not forget
that there are associated side effects.
So they're psychotomimetic, and as you can read all these here,
these are some of the side effects
that can happen with ketamine.
So it's important that -- it's a useful drug,
but people need to be familiar with the drug
and we cannot use just randomly.
And if -- it has received a lot of attention recently.
So if you do just ketamine and pain,
I tried to do that and I got like,
you know, I could buy ketamine here,
so you can buy for anxiety or depression
or anything like that online. And you can get it.
But also people like to make money and it's --
it has become really a big industry
with lot of side effects.
So as you can see it here, it has been on news.
So like initially, if you look at --
it's kind of interesting to see the timeline.
You would -- first you'll see all the benefits of ketamine,
then FDA issue of misuse of you know, ketamine and other thing.
And after Matthew Perry's death, it has become --
gained, like, a lot of attention on ketamine
and how to do it safely. So again, going back
to the American Society of Hematology guidelines.
So they gave it conditional recommendation.
And the reason for that is --
so they suggested it can be used for acute or chronic pain
in patients with sickle cell disease.
And the -- and here they also give the dose.
So it's used in the sub-anesthetic or small dose.
So it's generally 0.1, to 0.3 milligram per kilo per hour
and up to 1.
So that's, like, the maximum dose.
So Azure guidelines gives us that dose and again,
leaves some of the room for like physician discretion.
And those are acute pain that is refractory
in the treating physician's opinion.
So there are no --
and we don't have a definition of refractory pain.
So it kind of ends with the physician
when they want to try that.
They also make comment, it's important to do
safe administration of the medication,
and also only if the expertise is available at a institution
to do it safely.
So in conclusion, we all know that acute and chronic pain
is common in sickle cell disease.
Current treatment is opioid centric,
which is associated with many side effects
and may not be effective.
Cannabinoids are often used by our patients.
However, we don't have data on efficacy
and we don't know the risk and adverse effect
and especially its impact on the developing teen brains,
which are where the --
in this population, the use is increasing.
Buprenorphine, a mixed agonist and agonist
opioid receptor modulator,
it was shown to be effective in sickle cell population.
It was effective in reducing pain,
reduced acute care visit,
and also opioid use in small studies.
So all the studies I showed,
these are like small studies, open label.
Use is increasing in sickle cell disease,
but barriers still exist for its use.
Ketamine is -- can be used for chronic
or refractory pain and conditionally --
right now it's approved conditionally.
Recommended because of the limited data.
So we may need to have more data so that it can get --
and I talked about mostly, like, the pharmaceutical agent,
but as we heard earlier in the talk,
they're like whole area of the non-pharmacologic approaches
also that can be integrated in the care,
so we can avoid the side effect of different things.
That's it. Thank you so much for your attention.
Zachary Ramsay: All right. So thanks for that talk,
which is the last one for the session,
but we're going to welcome two quick questions if anybody has.
Male Speaker: Hi. Great talk. Thank you for the review.
So regarding cannabis, to the best of our knowledge,
does use -- how does use affect perhaps
the incidences of acute chest syndrome, right?
It's a type of smoking,
but at the same time you might be treating pain
that you could argue it can go almost back and forth
and obviously we're still at the research level.
So I'd love your thoughts on that.
And then number two, what are your thoughts?
Again, it's still -- we're still in the investigation stages,
but the different formulations of cannabis,
whether it's the synthetic forms or the derivatives
or even the different ways to deliver the --
I think there's different formulations these days,
so I'd love your thoughts on that.
Deepika Darbari: Okay.
So first of all, disclosure, I'm a pediatrician [laughs],
so I don't prescribe any cannabis
to any of my patient [laughs].
And second thing, I do want to say, like,
you know, my approach has been more like whole person care
because as a pediatrician, my goal is to make our patients
independent and in future they can support themselves.
So when I counsel them, I said, if I'm taking marijuana,
do you think my hospital will keep me employed?
And answer is no.
So what makes you think that you would be using?
But that's not your question.
So your question is like, you know, of what kind of --
how can we use it?
So the one trial that was used --
that used the inhaled vaporized,
but there was a side effect of like in that study of acute
and in the case --
so there were certain side effects,
smoking in general, marijuana or something else
we don't recommend for our patients
just because, you know risk of acute chest syndrome.
Because smoking is just bad.
So if our patients, if they are taking the compound or any --
so we would suggest like it's better
to take maybe edible than smoking.
So those are the really main thing.
And I don't think we have enough information
on synthetic versus you know,
natural forms of marijuana or any other drug.
And there was a article, I want to say maybe New York
Times, but they said if you think about
what people smoked maybe 30 or 40 years ago,
it's very different from what's available in market now.
So maybe the compound which was smoked by,
you know, older generation that was much more pure
and didn't have all these additive.
So it's kind of has become very dangerous
because they have certain other things
added to it for more effect,
and that's contributing to more of a problem.
So hopefully there'll be more research in this area,
but we don't have a lot of answer.
Yes.
Male Speaker: So do you know the number of recorded
overdoses of marijuana use?
Deepika Darbari: No, I don't know.
I mean, I can guess it's high, but I don't know.
Male Speaker: No, it's actually zero.
There are --
there are no recorded overdoses of marijuana use.
Deepika Darbari: Okay.
Male Speaker: And opioids are now at,
what, like, 110,000 deaths in the U.S. last year?
Deepika Darbari: Yeah. No, absolutely.
I'm not suggesting opioids are better or, you know --
you know, marijuana, which one is better,
but I'm just suggesting that we should think about side effect
and what's the person's goal is. You know, pain control,
there are other things that can be used for controlling pain.
That would be my -- especially for pediatrics.
Cheryl Stucky: Well, and I think specific to pediatrics,
so in our state where products were legalized,
we've seen in pediatrics an increase in toxicity
in like very young children who found the products.
So there are overdoses in that regard. So --
Deepika Darbari: Yeah, and that was the one
of the things like, for example, Colorado and other states,
they said if the stuff is legalized,
it'll reduce the use and mortality and accident.
So you may not die from overdose,
but you may be driving under the influence
and may get killed and kill others as well.
So --
Jane Little: Hey, Dipaki, that was great.
Deepika Darbari: Thank you.
Jane Little: It's Jane over here.
Jane Little. Deepika Darbari: Yes.
Jane Little: How do you integrate mental health
into this kind of view of different medications
that you use? Like do you bring it --
we try to bring it in very early,
but I don't know if that's universal.
Deepika Darbari: Yeah, so we have a --
we have this clinic called integrative clinic
or whole-person care where all the medications are discussed.
Obviously the first treatment is like sickle cell disease
modifying therapy along like hydroxyurea.
But all these things are offered.
So if, for example, ketamine is --
we use often in ketamine,
and mostly it's like patients who may have chronic pain
and admitted only in inpatient setting,
and there's a limit on how much can you use.
So not more than once a month, for example.
So those are the patient --
and not ketamine doesn't work for everyone.
So there are some patients who would like it,
but others don't want to try it.
Then for buprenorphine, we are using more now,
and those are typically the patients
who have been on high doses of opioid
and more like chronic opioid therapy.
But it's -- it -- mental health is so important
because many times pain is --
you know, because of the anxiety,
depression and some of the other things.
So if you don't correct those, then you know, it's --
it may not work.
So our approach has been a comprehensive approach.
We are lucky to have a psychologist in our --
in our clinic as well as working with a psychiatrist
who has this interest in buprenorphine
in sickle cell disease.
So we have been kind of bring it together, give --
educate our families and hopefully,
you know, they'll get better. Thank you.
Okay. Thank you so much.
Haydar Frangoul: Well, thank you everybody.
I have the pleasure of be chairing the next session
on the mechanism based therapies for sickle cell disease.
I would like to commend the previous session
because they stayed on time.
So my goal is to stay on time here.
So this session is what's keeping you guys away
from happy hour and dinner, by the way.
So we just need to keep it on time.
So it is my pleasure to introduce Dr. Daniel Bauer
who is the Donald Frederickson Associate
Professor of pediatrics at Harvard Medical School
and the director of the gene therapy program
at Boston Children's Hospital.
Dr. Bauer was not able to attend the meeting,
so he's going to deliver his talk virtually.
So I think they are going to queue it in.
Daniel Bauer: Hi, can you hear me?
Great. Here. Let me share.
All right. It's a bit of an echo for me.
Okay. Well, please let me know
if there's any problem technically,
and thanks Swee Lay and John and all for the opportunity
to participate in this terrific meeting.
I'm really sorry I couldn't be there in person.
I -- okay, great. I'm just looking at the chat.
Yeah. So anyway, I just came off hematology
inpatient service this morning.
And I think many of us are facing cognitive dissonance
where on the one hand we have this remarkable opportunity
to offer sickle cell disease patients
approved genetic medicine service.
We discussed and consider gene
therapy options role of our patients.
And actually we got input from Jane
Little whose voice I just heard.
On the other hand, the therapies are complex, costly, toxic,
and cannot scale to address the unmet clinical
need of an underestimated 300,000 babies
born around the world each year with sickle cell disease.
And so I would argue this means
we still need to develop better approaches
that are simpler, safer, and more effective.
Here's my disclosures.
So clinical observations dating back to those
from Janet Watson in the 1940s indicate that newborns
with sickle cell disease are protected
due to the high level expression of fetal hemoglobin.
Disease only begins once, fetal gamma globin,
the paralogue and genomic neighbor of adult beta globin
has been transcriptionally silenced after birth.
A longstanding goal of hematology
has been to deeply understand hemoglobin
switching to enable therapeutic induction of fetal hemoglobin.
When I joined Stuart Orkin's Lab as a hematology fellow,
I became interested in how common genetic variation
at BCL11A influenced fetal hemoglobin.
We observed that the disease associated in chronic
SNPs identified in Swee Lay's
pioneering study and validated many times since overlapped
an erythroid enhancer cloning signature.
We use gene editing to disrupt the autologous regulatory
sequences in a mouse erythroid cell
and observe that the erythroid BCL11A enhancers
were selectively essential for erythroid BCL11A expression.
Our CRISPR dense mutagenesis
where each dot shows an individual guide
RNA, identified critical minimal sequences at the BCL11A
enhancers that could serve as therapeutic targets.
We found GATA-1 binding sites at the plus 58
and plus 55 enhancers that were required
for robust fetal hemoglobin silencing.
So this led to a simple therapeutic vision to disrupt
the BCL11A enhancer
in hematopoietic stem cells leading to reduction of BCL11A
specifically in erythroid precursors and subsequent,
the repression of fetal hemoglobin.
Ex-vivo modified HSCs could serve in an autonomous graft
for hematopoietic transplant.
Recent clinical trials by CRISPR therapeutics
and Vertex have shown that in 43 sickle cell disease patients,
ex-vivo gene editing can produce efficient indels in blood cells
with about
70 fives edited potent fetal hemoglobin induction
comprising about 40 percent of total hemoglobin
improvement in hemolysis
and reduction in vaso-occlusive episode frequency,
leading to the FDA approval of CASGEVY in December, 2023.
Nonetheless, patients had some residual hemolysis
and six out of 43 had two episodes after therapy.
Long-term outcomes, including the impact
on the insidious deterioration characteristic of sickle cell
disease organ function remain unknown.
This study from Belisario et al.
reports a Brazilian cohort of Hb S/HPFH compound,
heterozygous children with mean hemoglobin about 12
and a half grams per deciliter and fetal hemoglobin
around 42 percent, which are similar values
as reported in the recent clinical trial
and revealed great amelioration of sickle cell disease,
but still some episodes of acute chest syndrome.
Acute pain episodes and transfusion requirements
suggesting clinical manifestations
of sickle cell disease were not completely reversed,
meaning individuals with sickle cell trait
with heterozygous HBS mutation are largely healthy.
Under venous oxygen tension of about 4 percent,
it takes about 65 percent adult hemoglobin to prevent sickling,
which is the precise distribution of adult hemoglobin
found in sickle cell trait.
50 percent hemoglobin will be expected
to achieve a similar degree of sickling protection,
suggesting that slightly more robust fetal
hemoglobin induction might yield clinical outcomes,
non-inferior to sickle cell trait.
Recent work from my lab led by Jing Zang
has shown that combined targeting of the two major
BCL11A erythroid enhancers plus 58
and plus 55 reduces superior enhancer disruption
compared to single enhancer disruption or alternative
approaches like BCL11A knockdown or gene
editing targeting the gamma globin promoter minus 115 site.
The combined BCL11A enhancer editing approach
produced the greatest fetal hemoglobin induction
and prevention
of in vitro erythrocyte sickling attributable to disruption
of both GATA-1 binding sites at both major enhancers up
to four total binding sites disrupted per cell.
With our colleagues John Tisdale and Selami Demirci at NHLBI,
we tested this combined enhancer gene
editing protocol in the rhesus macaque,
non-human primate where we could follow many of the same steps
as would be done in patients including mobilizing,
collecting and purify hematopoietic stem cells.
Prior to gene editing, ex vivo and autologous infusion,
we tested cohorts.
One was treated with standard busalan chemotherapy
and the other treated with an anti-CD45
antibody drug conjugate.
We observed similar high level gene edits
in the engrafting blood cells
and similarly robust fetal hemoglobin induction
with or without chemotherapy
over at least a year of follow-up,
providing proof of concept that chemotherapy
is not required for effective hematopoietic reconstitution
by the gene edited cells.
One possible risk of gene editing is off-target effects.
With our bioinformatic colleagues,
we built a computational tool called CRISPRme to predict
the impact of genetic variation on off-target risk.
We predicted that the most likely off-target effect
of the gene editing approach
is due to a genetic variant shown on the left in blue,
which creates a PAM sequence that allows Cas9 to bind
in an adjacent site at the CPS1
locus with just three mismatches.
This variant is found at about 5 percent allele frequency
in African ancestry.
Individuals suggest to one in 10 sickle cell
disease patients might carry this variant.
Since the off-target sites on the Q arm of chromosome
2 more than 150 megabases from the BCL11A target site, a P arm,
we hypothesized that simultaneous cleavage
of the sites could result in pericentric inversions,
which indeed we confirmed were present at about 1
in 600 allele frequency
after gene editing in donor hematopoietic cells
carrying a heterozygous risk variant.
We think genetic variant associated off-target editing
is a generic risk of gene editing
and should be considered in editing design,
patient screening and clinical follow-up.
Alongside its approval of cevy in December, 2023,
the FDA mandated a post- marketing surveillance study
to screen patients for this CPS1 variant
and assess for genetic variant associated off-target editing.
Many potential genetic variant associated off-target effects
are due to much rarer variants than the CPS1 variant,
which might make it infeasible to find donors
for experimental testing.
Here I'm showing the minor allele frequency
of 72 candidate variant off-targets
based on cutting frequency determination or at least 0.4.
Most associated with very rare genetic variants.
It would not be feasible to obtain CD34 cells from donors
carrying each of these variants
for direct off-target assessment.
So to address this challenge, we've developed an assay
that can assess these variant off-target edits
while maintaining clinically relevant cellular
and gene editing delivery context.
The experimental design of our so-called ABSOLVE-seq assay
involves using lentivirus to introduce alternative
allele sequences into the 34 cells
and then performing editing following
clinically relevant protocols. We include unique molecular
identifiers to detect single editing events
and we test a pool of targets together in a single experiment
within an accompanying computational pipeline.
Here are the results of our pooled experiment.
I'll summarize a lot of data
that we do find the three expected targets,
the two on targets in this experiment,
and one variant associated off-target.
And with unprecedented sensitivity,
we do not detect off-target editing
at each of the other 72 candidates
providing reassurance of the sensitivity of our approach.
So these data helps support our clinical trial application,
which has now been FDA cleared
and anticipate beginning enrollment
at Boston Children's Hospital in early 2025.
More broadly, this demonstrates a scalable strategy
to tackle off-target assessment
for novel candidate gene editors.
So we have great opportunities ahead for sickle cell disease
therapeutic gene editing.
With genetic medicine, we can aspire to make a large impact
on the clinical unmet need.
And due to the unique accessibility
of the blood system,
we can expect to deeply understand
the impact of gene editing at molecular,
cellular and physiologic levels. With indefinite follow-up.
I think we have a responsibility to our patients
to follow their outcomes closely and to develop
improved therapies to provide a safe,
effective and accessible therapies as possible.
I'm extremely grateful to current
and former lab members, mentors, colleagues, collaborators.
There's -- and especially sickle cell disease patients
and families. Thank you.
Haydar Frangoul: Thank you so much.
We have a time for a couple of questions.
Any questions for Dan? No? All right. Okay.
Well thank you so much for the presentation.
You did a lot of legwork for my presentation.
Thank you.
Daniel Bauer: Thank you.
Swee Thein: So, Haydar -- oh, there you go.
Let me introduce you.
Haydar Frangoul: Okay, that's fine.
Swee Thein: So I'm really delighted
that Haydar Frangoul could join us today
and he's going to talk to us about induction
of fetal hemoglobin using genetic approaches.
Haydar is the director of the pediatric stem
cell transplant program at the Sarah Cannon Research
Institute and Tristar Centennial Children's Hospital
in Nashville, Tennessee. So thank you so much for coming.
Haydar Frangoul: Thank you so much for inviting me.
Thank you for the organizing committee
to invite me to give this talk.
I think this is -- is this how you --
the other -- the other one.
Okay, perfect. Okay. These are my disclosures.
So again, as mentioned by Dan earlier,
that symptoms of sickle cell disease
start after the switching in the main type
from hemoglobin F in the prenatal life
to adult hemoglobin in patients with sickle cell disease.
The onset of symptoms
occurs as hemoglobin F switches to hemoglobin
A, which is affected by disease-causing mutation.
I'm not going to spend a lot of time on this
because this is very well known to everybody.
So this is a paper by oral plat in 1994
following 3,700 patients in the NIH cohort for several decades.
And the observation in that paper was that individuals
who maintain a fetal hemoglobin of over 8.6 percent over time
have almost doubled the survival by age 50
as compared to those who do not maintain high levels
of fetal hemoglobin long-term.
So a subset of patients with sickle cell disease
who continue to express fetal hemoglobin into adulthood,
a condition known as persistent fetal hemoglobin experience
less symptoms.
And usually by the time you reach a fetal hemoglobin
of around 30 to 40 percent,
the patients become relatively asymptomatic.
So genetic studies as mentioned by then earlier,
this described the genetic variation
leading to decrease expression of BCL11A results
in increased hemoglobin F expression.
So BCL11A became a very important target
in designing genetic therapies to induce fetal hemoglobin.
So there are multiple trials which I'm going to go through
within the limitation of time we have.
There is the paper reported using a antiviral approach
by the Boston Children Group.
There is the CRISPR-9 gene editing of the HBG1,
HBG2 promoter that was run by Novartis.
I'm going to go into more details in the two trials,
which is the exa-cel trial
leading to CRISPR being approved.
And I'm going to show you new data
that we just presented in IHA June and reni-cel,
which uses Cas12A editing of the HBG1 and HBG2 promoter.
And also there is data that was recently presented
a couple of months ago that I'm going to present here.
And there is another trial ongoing, which is the BEAM-101,
which uses a totally different approach,
which is base editing to introduce naturally occurring
A-to-G substitution into the promoter of HBG1 and HBG2.
Now, BEAM-101 does not have any publicly available data,
so I'm going to limit my presentation
to the top four studies.
So this is the paper by Dave Williams Group
from Boston Children
and they use BCH-BB694 antiviral vector,
which encodes a short hairpin RNA
targeting the BCL11A messenger RNA,
embedded in the micro RNA,
allowing erythroid lineage specific breakdown.
The paper reported on six individuals
with sickle cell disease treated
and all of them achieved the fetal hemoglobin
between 20 and 41 percent with only three patients
having fetal hemoglobin greater than 30 percent with F cells
ranging between 58 to 93 percent.
And following this therapy,
none of the patients experienced any vaso-occlusive crisis.
This paper came out in 2021.
The next paper came out in 2023, which is by Akshay Sharma
is the study using CRISPR-Cas9, editing of the HBG1,
HBG2 gene promoter that treats sickle cell disease.
This is based on basic science work
that showed that targeting this promoter region
can increase fetal hemoglobin in animal love in animal studies.
And they screened multiple guide RNA
and ended up with a guide,
RNA number 68 that they used for this study.
And there were three subjects treated,
reported in New England Journal of Medicine last year,
a total hemoglobin at last follow-up range
between 10.4 and 11.9.
And the fetal hemoglobin at last follow-up was 19
to 25 percent of the total hemoglobin.
So the patients continued to be slightly anemic
and the fetal hemoglobin was less than 30 percent,
and all patients had some clinical benefit
from this therapy.
My understanding is this trial is not accruing anymore.
I want to spend some time talking about the exa-cell data,
which then refer to --
resulting in the approval of CRISPR last December.
So exa-cel mechanism is grounded in human genetics.
It is basically targets the BCL11A
erythroid enhanced region,
resulting a reactivation of hemoglobin F
and the infusion of exa-cel, as I'm going to show you,
resulted in pancellular hemoglobin F production
with a fetal hemoglobin of around 40 percent
resulting in significant reduction
or elimination of VOCs.
So the patient journey is relatively complicated.
This is not easy studies to do, or easy therapy to deliver.
It takes a long time.
Patients are -- usually undergo --
exchange transfusions for around four to eight weeks
prior to peripheral blood stem cell collection
stem cell collected using plerixafor.
And because we use plerixafor the mobilization
is not as robust as when you use GCSF,
which you cannot use in patients with sickle cell disease cells,
and then manufactured and then shipped back
to the transplant center
where the patient undergo myeloablative chemotherapy
with busulfan which is pharmacokinetically adjusted.
The cells are infused
and the patients are followed for two years.
This was a phase three multicenter trial,
that enrolled patients between the ages of 12 to 35.
And they define severe disease
as having two vaso-occlusive crisis per year
in the last two years.
So basically four in the last two years.
The primary endpoint was being hospitalization.
The primary endpoint was being VOC3 for 12 consecutive months.
And a secondary -- key secondary endpoint
was being hospitalization 3,4,12 consecutive months.
And that's HF12. And because of the trial,
met the primary and secondary endpoint,
which I'm going to show you a little bit later.
It was as mentioned previously,
the product was commercially now available
and approved in the United States,
the EU, the United, Kingdom,
Kingdom of Saudi Arabia and Bahrain.
So the full dataset is for all 46 patients.
Now the primary efficacy is the 39 patients
that have long enough follow-up.
The mean age was 21 years of age.
They were equally divided
between male and female subjects.
The majority had beta as beta as genotype.
And of note, around 40 percent of the patient
had one or two alpha gene deletions.
And the number of historic VOCs were four per year
in the last two years.
And the median number of hospitalization
was almost three per year in the last two years.
So the patients required the median mobilizations of two,
but ranging from one to six.
The number of CD34 cells infused was 4.7 million,
CD34 per kilogram,
and the median duration of follow-up was 28 months.
All patients engrafted, neutrophil and platelets
at a median of 27 and 34 days respectively.
And of note, for those of you
who have used single agent busulfan
and there is really around nine days after infusing that cells,
the patients have normal counts.
So the duration of neutropenia was only 17 days.
And the time to the last red blood cell
transfusion was 19 days,
and the hospital stay was 30 days.
So this is a complicated graph here.
I just want to orient you.
This is a swim plot looking at the number
of vaso-occlusive crisis pre and postsl infusion.
I think I have a pointer here, maybe.
Yes. So the area here in gray represent two years
prior to the exa-cel infusion,
and each diamond here represent a VOC vaso-occlusive crisis.
The red dark bars are the times
when the patients were receiving red blood cell transfusion,
and the dark gray is 60-day washout.
So the clock starts for the efficacy from 60 days
after the last transfusion.
So 36 of the 39 individuals on this trial,
or 92.3 percent achieved the primary endpoint
of being vaso-occlusive 3 for 12 consecutive months.
And the average duration range from 12 months
to around five years.
Seven participants who are in the bottom,
who are not included in the primary efficacy,
these are patients who don't have long enough follow up.
There are seven of them and five are still eligible
to meet the VF12 definition.
Pain events happened after infusion of exa-cel,
and it usually was associated with either infection
like parvovirus B19, influenza B or COVID-19,
or a procedure like a bone marrow biopsy
or the use of corticosteroids.
More importantly, the patients who experienced VOCs
after exa-cel also had robust increase in fetal hemoglobin.
This is the secondary -- this is -- this figure here
divides the patient between adults and adolescents.
Adolescent defined at 12 to 18 years of age
and 92 percent of the adults
and 90 percent of the adolescent patients
achieved the vaso-occlusive 3,4,12 consecutive months.
This is the key secondary endpoint
looking at hospitalizations, again,
to orient you the area here on the left
to present the hospital admission.
Each dot is one hospital admission
and 38 of the 39 evaluable patients,
or 97.4 percent met the secondary endpoint
of being hospital free for 12 consecutive months.
And the mean duration of hospital
free ranges from 12 months to almost five years.
This, on the left here shows the hemoglobin percent,
and this is divided between adolescent and adults.
The adolescents are in black, the adults are in blue.
And as you can see,
the mean hemoglobin F was 45 percent at one year
and 42 percent at two years in the adolescent.
And very similar in the adult cohort.
The graph on the right represent the total hemoglobin,
and again, the mean hemoglobin in adult at --
in adolescent at one year was 13 and at two years was 13.2.
And the mean hemoglobin in adults was 12.7 and 13.2
at one and two years respectively.
So the majority of the patients had normal
or near normal hemoglobin.
When you look at the distribution of fetal cells
red blood cells containing fetal hemoglobin,
the F cells, more than 95 percent of the cells
contained fetal hemoglobin by month three.
And this was sustained over time.
Actually, those dots represent a standard deviation,
which is very tight, so that's why you can see the bars.
We evaluated the editing percentage
in peripheral blood and bone marrow.
The top panel represents the bone marrow.
We looked at CD34 cells and looked at the percent
editing in stem cells in the bone marrow,
and we looked at whole blood nucleated cells in the bottom.
The green lines represent
those who achieved the primary endpoint.
The red bars took about the patients
who have not achieved it,
and you can see the editing percentage
was very stable and durable over time,
both in the bone marrow and peripheral blood.
When you look at hemolysis,
which is very important in sickle cell disease,
intravascular hemolysis looking at LDH and haptoglobin,
LDH was elevated and haptoglobin was very low at screening.
And both of those either normalized
or became very close to normal after infusion of exa-cel.
The reticulocyte count dropped and the indirect bilirubin
improved quite dramatically after exa-cel infusion.
We have done patient reported outcome.
It's not only important to see
if we have elevation in fetal hemoglobin,
it's also very important to see if we actually --
that resulted in patient benefit
and we looked at patient reported outcomes.
This data was presented at EHA in 2024.
I'm just going to show you a few graphs from it
for the sake of time.
This is looking at a tool called FACT-G,
which looks at the total scores in adults.
And as you can see,
there was dramatic improvement from baseline
to last follow-up 36 months.
Similarly in the another tool called BMT scores in adult
in primary efficacy point,
and then there was significant improvement
in quality of life over time.
When you look at the pain scale,
there was a significant drop in reported pain from baseline
to last follow-up. So the side effects.
So this therapy is not without side effect.
I keep telling John he needs to find a way
to treat patients without busulfan.
So all the side effects you see here are related
to the conditioning regimen with busulfan.
So, as you expect with high dose chemotherapy
and autologous transplant,
patients experienced dermatitis, nausea, fever neutropenia.
More importantly, there were no secondary malignancies
and no serious adverse events related to exa-cel.
Unfortunately, one of the individuals
enrolled on this trial
passed away related to COVID-19 infection
and respiratory failure.
Just of note, this patient was not immunized
and the trial was performed during the COVID era.
We were very careful trying to protect patients from COVID,
but this was an unfortunate event.
So in conclusion, for the exa-cell data
is the first and only approved
CRISPR-Cas9 gene editing therapy.
It resulted in 92.3 percent VOC
free rate and 97 percent hospital free rates.
There was durable hemoglobin F that lasted now up to five years
with the longest follow-up.
There is stable allelic editing in the bone marrow
and peripheral blood that was also durable.
And the safety profile is very consistent
with what we expect with busulfan.
And again, this could be have the potential
to be a functional cure
for patients with sickle cell disease.
I want to profess here,
although I am the lead investigator on this trial,
is that we still don't have real long enough data,
as Dan mentioned earlier,
to determine if this therapy will affect
really the end organ damage 15 years down the road.
So that's why the patients are all enrolled
on the long-term study for up to 15 years.
Now, switching gears to the editors clinical trial,
which is reni-cel which uses a proprietary Cas12A
and they're editing the HBG1 and HBG2 promoter region.
You saw data in the Novo Nordisk data using CAS9.
This is used as CAS12,
which is more specific to that site and more effective.
And the idea is to induce fetal hemoglobin similar inclusion
and exclusion criteria to the exa-cel trial.
Except this trial includes patients up to 50 years of age
while exa-cel stopped at age 35.
They defined VOC again as severe VOC
as two events per year in the last two years.
This -- there was screening mobilization,
apheresis, manufacturing, and infusion
identical to the trial, I presented earlier.
There were 18 patients reported this June and IHA.
And this is the data that was provided to me
by Dr. Hanna and the sponsor.
Most of them had a beta, as beta as genotype,
and the median age was 27 years.
And the median number of VOEs in the previous two years was five.
And the number of mobilizations were similar to exa-cel of two,
ranging from one to four.
The number of CD34 infused were 4.6 million per kilogram.
And the follow-up was seven months.
At the time, all patients engrafted neutrophil
and platelets at 23 and 24 days respectively.
Looking at the outcome here, although we don't have
a lot of patients on long follow-up
all patients after infusion of reni-cel remained VOC
free for up to 22.8 months.
And the study continues to enroll and accrue patients.
This is data describing the total hemoglobin
as well as the fetal hemoglobin.
You can see by month four,
the fetal hemoglobin was over 40 percent
and it was sustained over time.
The hemoglobin reached normal levels by month three
and was sustained over time.
All hemolysis markers, including reticulocyte,
indirect bilirubin, LDH and haptoglobin,
either improved or normalized after infusion of reni-cel.
Again, the F cells are quite high indicating
a very uniform distribution of the fetal hemoglobin
and red blood cells, which was over 95 percent.
And they also performed editing evaluation
in the bone marrow and blood, which was sustained over time
and high in the stem cells in the bone marrow.
When you look at side effects, again, all the side effects
are related to the high dose chemotherapy
and autologous stem cell infusion basically stomatitis,
and fever neutropenia.
So all of them were related to busulfan.
There were no serious side effects related to reni-cel.
So in conclusion, reni-cel again
is another product investigational autologous gene
editing medicine that demonstrate promising results.
In gene editing, it showed that the patient remained VOE free,
all patients experienced in early correction of the anemia
with sustained high fetal hemoglobin over time.
And the treatment for reni-cel showed favorable safety profile
and promising preliminary efficacy
supporting the further investigation.
The trial now is, actually accruing
and treating adolescent cohort.
I just wanted to show you a couple of things.
This is a smear since everybody was showing pictures,
so I like to do that too here. I was jealous.
So this is a smear from one of our subjects
that enrolled in our site and the exa-cel trial.
This was actually a patient who was not having any crisis
when I screened them.
And this is their peripheral smear at enrollment.
This is their smear at one year post transplant.
Then I would like to show that one of our patients,
this was reported yesterday in the news.
So I modified my slides today.
This is our patient, Jimmy, that was reported in the news.
He was the first sickle cell patient to climb Kilimanjaro
and make it there and come down.
He just texted me and he is back in Atlanta doing very well,
and he was able to do it.
He'll be the first patient to climb Kilimanjaro.
I would like to thank first and foremost the patients.
This is Victoria Gray, which made a lot of news nationally.
She is the first patient to enroll.
When I consented her, I said,
we are giving you a CRISPR modified stem cells
that nobody has ever received. We don't know if it works.
I have no clue if this is going to even help you long-term.
And she said, sign me up.
So she was the first one to do it,
and thankfully I just saw her for her five year follow-up
and she's doing extremely well. She was not able to work.
Now she's working full time and taking care of her kids.
This is -- I would like to thank our team.
This is January of 2024, we --
when we celebrated our 40th infusion
of genetically modified,
genetically edited stem cells in patients
with sickle cell disease and beta thalassemia.
This study would not have been able to be conducted in our site
without an entire village. Thank you.
I'm happy to take any questions.
Swee Thein: Thank you so much. That was a really good overview.
So I see one hand raised. In fact two.
Maybe let's start with Mark Walters.
Haydar Frangoul: Oh, I'm in trouble.
Mark Walters: Hi, Haydar. That was really --
I think both those approaches using the CRISPR technology
clearly established the efficacy of the treatment
certainly in the short term, if not the long term.
So my question has more to do with safety and we know that,
that double strand of breaks in true hematopoietic
stem cells elicit untoward effects and cell death.
And from David Kent's careful work
characterizing six patients in David Williams' trial,
that reconstitution of hematopoiesis can --
occurs in as few as several thousand true hematopoietic
stem cells in the lentiviral approach.
I predict it's even fewer after CRISPR disruption.
So what can you tell us about clonal hematopoiesis
after these treatments? Have you --
been have you been following the natural history
of this post-infusion?
Haydar Frangoul: The short answer is no.
None of these studies really did clonal hematopoiesis.
And this was a decision made by the sponsors.
Now we are performing bone marrows routinely
and we are storing bone marrows for testing
if something is to arise in the future.
But clonal hematopoiesis,
I think the point you're raising is very important.
And this has not been studied very well in this setting.
Female Speaker: Yeah. It's over here.
Yeah, that was a fascinating talk.
In the exa-cel trial, you measured pain
and you saw that that went down in the patients.
Was that presumably that was chronic pain
because they're not --
are they not having any acute pain whatsoever
because they don't have VOEs and then -- yeah.
How did you measure the pain?
Haydar Frangoul: Yeah. Well, this is a great question.
So the majority -- actually two thirds of the patients
in the exa-cel trial were adults.
And I can speak on which a lot of the patients
were from our side.
A lot of patients came to us as adults with, you know,
AVN in the shoulder, AVN in the hip,
that they were on chronic opioids.
I can tell you within our side data,
which we have not yet really,
truly looked back at the pain scale
and looking at which is chronic,
which is acute, is everybody in our site
was able to come off their chronic opioids.
Now, several individuals had to go back
and get their hips and shoulders replaced,
which they were planning pre,
but they postponed it until after
and they were able to come off the medications.
But as you can tell -- that, you know, sickle cell disease,
as you know, is a chronic disease
that leads to a lot of long-term complications.
So a lot of those damage joints
are not going to get miraculously better
because we infused exa-cel. Correct.
So I think there is a component of chronic pain.
And in fact, I think also what is --
was an eyeopener is even with Victoria
in the first six months afterwards,
she calls me and says, doc, I think my sickle cell is back.
It's like, okay, so Victoria, what's happening?
It's like, well, my legs hurt.
What did you do? I cleaned house all day.
I worked, I took my kids to school
and what is a baseball game.
Well, I'm hearing that I am hurting. Correct.
So I think there is also a lot of anxiety
in the patients about pain
and attributing pain from daily life.
Also, I think that issue really needs to be addressed
not only in the gene therapy but in the transplant setting
to see what happens to pain afterwards.
I think this is a huge need in the field.
Female Speaker: That's fascinating.
Thank you so much.
Swee Thein: So you have one comment
from one of the people listening hybrid online.
It's a groundbreaking innovation. Congratulations.
Haydar Frangoul: Well, thank you.
This is a team effort. This is not only me. Thank you.
Swee Thein: I want to ask you one last question.
Yes, sure. In one of your slides,
the person we followed,
I think the longest, hemoglobin reached 17.6 total.
Haydar Frangoul: In the reni-cel?
Swee Thein: That's the reni-cel. Yes.
Haydar Frangoul: The reni-cel. Yes.
The hemoglobin was high and actually it had --
it's having no adverse event on the patient.
The patient is doing very well, but you are right,
there were occasional patients even on the exa-cel trial
that reached the hemoglobin in the 16 year range.
Swee Thein: So, and there are no issues.
Haydar Frangoul: No. No issues, no clinical issues
at least that we can see from the increased hemoglobin.
Male Speaker: Oh, yep.
Swee Thein: You are going to take over.
Haydar Frangoul: Yep. I am going to take over.
Thank you. So I'm going to --
I'm going to introduce our next speaker.
Oh, there's another question. Yes.
Male Speaker: Sorry, I just had --
Haydar Frangoul: Yeah, sure.
Male Speaker: Yeah. So thank you for your talk.
So similar to Mark's question in double strand breaks,
comparing the reni-cel and exa-cel data, right?
There are some differences in media
and neutrophil engraftment times
and you're looking at the ranges.
Can you comment all that? We don't have any --
at least I don't think public data on GCSF
use post-transplant.
Right. Just trying to compare those engraftment times.
Can you speak to GCSF use?
Haydar Frangoul: I don't know the GCSF use
for individual patients.
I know that the studies allow GCSF use
if there is no engraftment
at the discretion of the investigator
by day 21 with consultation with the medical monitor.
So I know that some patients received GCSF
just because they had an ASC of 400 and they were bored.
They wanted to leave the hospital,
so they needed to meet engraftment.
So they received GCSF to engraft.
But I don't have the exact data,
but I know both studies allowed the use of GCSF after day 21.
Male Speaker: Thank you.
Haydar Frangoul: Oh, you're welcome.
All right. It's my pleasure to introduce the next speaker.
The talk is the induction of fetal hemoglobin,
small molecule and pharmacological approaches.
It is Dr. Scott Peslak.
Dr. Peslak is a physician scientist
and an assistant professor of medicine
at the University of Pennsylvania
and cares for patients
at UPenn Comprehensive Sickle Cell Program
and comprehensive adult thalassemia program.
Thank you.
Scott Peslak: Great. Thank you.
I'd love to thank the organizers for inviting me
to speak here today. It's my first time in Jamaica.
It's really lovely and it's really fantastic to hear
all this wonderful research going on here in the field.
So as Dr. Frangoul said,
I'm an adult hematologist at UPenn
and I see sickle cell disease and thalassemia patients.
I'll be talking today about small molecules
and pharmacological approaches to inducing fetal hemoglobin.
And these are my disclosures.
So we all know -- what we're all here for
is a study of sickle cell disease.
And the point I wanted to make with this slide
is that we not only want to treat acute complications
from sickle cell disease, but really to prevent these
from happening in the first place by using disease
modifying therapies to prevent occlusive pain episodes
and prevent chronic complications,
which can really occur in any organ system
throughout the entire body.
And we'll hear a lot about this tomorrow
in a whole series of talks
involving specific organ complications
in sickle cell disease.
There's been a tremendous amount of progress
on the pediatric side in improving mortality.
We heard this morning in sickle cell pediatric patients,
and this coincides with disease
directed therapy like hydroxyurea
as well as early treatment of infection and vaccination.
But we really haven't made a tremendous amount of progress
in the adult side. And so we --
and this is a study done by Lanzkron a few years ago,
and many others have looked at this as well,
that we need to move the needle
more in improving morbidity and mortality
and lengthening life and improving quality of life
in our adult sickle cell population.
So as you just heard from Dr. Frangoul,
there's been a tremendous amount of excitement
involving gene therapy as well as bone marrow transplant
[laughs]. Thank you.
And these really are transformative therapies
for the treatment of sickle cell disease.
However, there are some limitations.
This is challenging to deploy worldwide,
not only because of cost,
which we is a barrier we can overcome,
but also the expertise that's needed to actually be able
to deliver an autologous stem cell transplant to patients.
As Dr. Frangoul mentioned as well,
busulfan is not really a great conditioning regimen.
We need better conditioning regimens since this does
potentially result in things like secondary malignancies
as well as infertility in our patients.
So there's been a tremendous amount of efforts
trying to find new pharmacologic therapies
for the treatment of sickle cell disease.
And in the United States there's four FDA approved
therapies, hydroxyurea, which I won't talk about today
because there's a whole session tomorrow
focusing on hydroxyurea as well as Crizanlizumab,
which is a P selected inhibitor, Voxelotor,
which binds high oxygen and affinity
binding hemoglobin state
and now L-glutamine, which reduces oxidative stress.
And these work well for many patients,
but they don't work well for all patients,
particularly on the adult side.
So we need more effective pharmacologic options
for treatment of our sickle cell disease patients.
And what I'll talk about today
is the induction of fetal hemoglobin
from a pharmacologic perspective.
So you heard from Dr. Bauer and Dr. Frangoul
about this hemoglobin switch that occurs in --
during the perinatal period in which you have a switch
from fetal hemoglobin to adult hemoglobin.
And if we can reverse this switch,
the idea is that we can reverse a lot of the pathophysiology
and the complications that happen
in sickle cell disease patients.
And the reason this is the same figure
that we know from a few years ago now,
that either if you see induction of fetal hemoglobin
by hydroxyurea or by naturally occurring genetic variants
termed hereditary persistence of fetal hemoglobin,
these patients live longer and they do better
and have fewer complications.
And this is because cells that express fetal hemoglobin
that termed F cells are less likely to undergo vaso occlusion
and also less likely to hemolyze.
So if we could increase F cells, we really could reverse
all the underlying cellular pathophysiology
of sickle cell disease
and all organ systems can benefit.
There's been a tremendous amount of work
in the last 20 to 30 years,
involving trying to understand how this switch works
and the direct transcriptional repressors of fetal hemoglobin.
And again, Dr. Bauer explained this earlier work
that was originally described by Swee Lay Thein
and subsequently by Stuart Orkins
Lab, Dan Bauer, [unintelligible] and many others,
identifying BC11A as a direct transcription repressor
of fetal hemoglobin in addition to LRF
or ZBTB7A and NF1A and X,
which is identified by Garrett Lobo's Lab a few years ago.
And ideally, we'd like to be able to target these directly.
And this is what the therapy for -- gene therapy for --
as we just heard for BC11A is the target for.
However, from a therapeutic and pharmacologic perspective,
this is very challenging to target these transcription
factors directly with small molecules.
Thus my goal since I've been working in the lab,
first in Garrett Lobo's Lab as a postdoc and now my own lab
as well as others in the field to try to understand
what are the upstream of these regulators?
What can we target to be able to modulate fetal hemoglobin
in a positive way?
And we've had a tremendous amount of success
doing this with the collaboration with Junwe Shi
at the University of Pennsylvania,
in which we use CRISPR domain focus,
CRISPR knockout screens, where we take either two cells
which are erythroid cell lines
that are really good for you doing genetic studies
and infect them with specific guide RNA libraries
that target specific kinds of functions in the cell.
We can then differentiate and sort these
into fetal hemoglobin high and low populations
and identify which genes when depleted lead
to higher levels of fetal hemoglobin.
And these are some of the kinase
or some of the libraries we've targeted here,
I'll talk about today,
identifying many different targets
that have really expanded our knowledge
of how fetal hemoglobin is regulated,
not just at a direct transcriptional level,
but upstream of this as well.
And this really has broadened our idea of how switching works.
This is a really nice review,
written by my colleague Eugene Khandros,
in the Children's Hospital of Philadelphia
and Garrett Lobo that looks at both gamma globin,
repressors and activators. And this all,
there's a lot of lines in this thing in this graph.
So I'm not going to go through every one of these.
But I'm going to talk about a subset of these
that I think are important to highlight some of the advances
that have been made in the last five to 10 years or so here.
So the first I'll talk about
is something that came out of Garrett Lobo
and work that I've done as well,
identifying a regulator of fetal hemoglobin
called Heme-regulated inhibitor, HRI.
And this is one of the first libraries we've screened.
We looked at kinase screens libraries
because they're purportedly the most easiest
to be able to target.
And we depleted HRI and we saw increases in fetal hemoglobin
in our in vitro systems.
And we -- I'd elucidated the mechanism of this proceeding
via intermediate called ATF4.
So ATF four is translationally regulated by HRI.
ATF4, we showed binds to the minor enhancer,
the plus 55 enhancer, as you heard
from Dr. Bauer of BC11A and drives the transcription.
So if we deplete HRI, we can decrease
ATF4 and decrease transcription to BC11A
and thus increase levels of fetal hemoglobin.
And I showed subsequently that we can combine HRI depletion
with other modulators of fetal hemoglobin inducers,
including several I'll talk about today,
such as pomalidomide and UNC0638 to reduce --
to significantly increase levels of fetal hemoglobin by HPLC,
which is our clinical gold standard and reduce cellular
sickling in a synergistic and in additive way.
Another screen,
another target that came out of our initial screens
is a molecule called ZNF410.
And this emerged as a positive hit
in one of our CRISPR screens as well.
This is work led by-- in Garrett Lobo's Lab as well,
involving if we deplete ZNF410,
we can significantly increase levels of gamma globin.
And this works by modulating levels of CHD4.
So ZNF410 represses fetal hemoglobin and modulating CHD4.
Very intriguingly, CHD4 is the only direct functional target
of ZNF410 and erythroid cells. It's remarkably specific.
So this work came out of John Jang Long in in Garrett's Lab.
And at the same time, Dan Bauer's group published
nearly identical results, with the target of CHD4 that,
the ZNF410 bound regulatory elements completely account
for the effects of ZNF410 on fetal hemoglobin repression.
So it comes up with this model
where there's this genome wide unique
and really conserved set of motif clusters upstream of CHD4
that drive its transcription.
And by knocking this out, we can decrease CHD4
and potentially increase levels of fetal hemoglobin
in future treatment of sickle cell disease.
Another interesting study that came out of Mitch Weiss's group
at St. Jude identified a HIF1-alpha and VHL
as a modulator of fetal hemoglobin.
They show that VHL loss leads to fetal hemoglobin induction
via stabilization, interestingly of HIF1-alpha upregulation.
So we see here that targeting directly a VHL leads
to increases in gamma globin,
and in doing it does so by stabilizing HIF1-alpha
in a HIF1-alpha dependent mechanism.
If we knock out HIF1-alpha, we actually lose that induction
of fetal hemoglobin that's seen.
And interestingly, this actually works by promoting
direct interactions
between what's called the locus control region,
which is the super enhancer that's present
in the beta globin locus and the gamma globin promoter.
And as by a series of capture C studies that are shown here
in which we have a model -- at high oxygenation,
you have HIF1-alpha degradation, but with low oxygenation,
HIF1-alpha is actually stabilized and promotes
these contacts between the LCR
and the gamma globin gene region.
So Mitch's Lab asked, can we actually induce this
by stabilizing HIF1-alpha using a class of compounds
that's being studied in anemia of CKD
called prolyl hydroxylase inhibitors?
And indeed, if we treat cells in vitro with prolyl hydroxylase
inhibitors you see stabilization of HIF1-alpha.
That's, again, HIF1-alpha dependent with a knockout
and is directly correlative with induction of fetal hemoglobin
indicating this might be an evolutionary conserved way
to regulate fetal hemoglobin,
but also might lead to ways we can modulate this
using small molecules
to increase levels of fetal hemoglobin
in sickle cell disease.
One of the most interesting studies that came out
was presented at plenary ASH this past year
and as a, just published in science a few months ago,
is a study done by Novo Nordisk that looked -- using --
essentially, using a screen of cell bond degraders,
cell bond -based degraders of transcription factors,
trying to identify how can we directly target transcription
factors using small molecules.
And so they did a screen looking at fetal hemoglobin
and then Y axis and cell counts, the X axis.
You want to be able and essentially to identify targets
that allow for induction of fetal hemoglobin
without affecting viability.
And they identified this compound C,
which remarkably is very specific
for the degradation of the transcription vector
called whiz which is targets and depletes this
at a very low concentration.
They then went to modify and optimize this compound
and actually tested this in vitro
and in vivo xenotransplant mouse models
as well as in non-human primates,
and showed that they saw dose dependent increases
in gamma globin transcript
as well as increases in F cells and reticulocytes.
And mechanistically, it's still a little bit unclear
how this works but what they propose in the paper
is this actually might support repressive
methylation of chromatin and might actually be modulated
through the EHMT1/2 repressive complex.
So more common mechanism, but really interesting
that we can do a screen where we can actually identify,
directly identify molecules
that are involved in fetal hemoglobin regulation.
In terms of EHMT1/2 methyl transferase inhibition,
this actually would published a few --
excuse me, a few years ago at the same time
by Ben Ebert's Lab
as well as Jeff Miller's group using a compound called NC0638,
in which they saw dose dependent increases
in fetal hemoglobin levels
following either EHMT1/2 or G9a inhibitions same complex.
And this actually acts by increasing or de-repressing
the gamma globin gene region
and increasing context between gamma globin
and the locus control region.
And so you switch from more of a adult-like state
to a fetal like state
and might serve also as a modulator of fetal hemoglobin
in sickle cell disease.
And there are on ongoing large animal studies for this as well.
One of the most further advanced compounds that's being studied
currently is a compounds being developed
by Fulcrum Therapeutics in the inhibition
of the Polycomb Repressive Complex 2,
or PRC2 called FTX-6058 or pociredir.
And by inhibiting PRC2,
they've shown you can increase levels of fetal hemoglobin
and there's labs that are currently studying
how this is specifically working mechanistically.
There's some of the clinical trial data
presented at EHA this past year,
an ongoing Phase 1b study called the PIONEER Study,
where they dosed patients up to 12 milligrams daily
for 12 weeks,
and they saw a dose dependent increase in fetal hemoglobin
as well as a significant increase in fetal
hemoglobin from baseline change.
So we're eagerly rating the additional data
from this likely to be presented at ASH this year
and how this affects patients at higher doses
and whether there's any off target
or other other side effects with this compound as well.
One of the studies that is interesting in
a different methodology
is actually to be able to deplete repressive
co-repressors of BCL11A including DNMT1.
So it's been known for a while that molecules like decitabine
can actually decrease DNMT1.
And this study, this phase 1 study in sickle cell patients
actually combine this with tetrahydrouridine,
which inhibits Cytidine Deaminase and potentiates
the effects of decitabine in repressing DNMT1.
And so this is a study that looked at this decitabine
THU combination drug given twice a week for eight weeks
and increasing doses.
And in this they saw significant increases,
especially at the higher dose in fetal hemoglobin levels
as well as F cell number.
And there's a current ongoing trial called the ASCENT1 trial
using this looking at 1-to-1 ratio of either
once weekly, twice weekly or placebo
in terms of F cell induction using this compound.
So more to come on this in the coming in --
the coming year or two.
Two more compounds I wanted to talk about.
And then I want to talk about something a little bit of work
from my lab as well. There's a set of compounds
looking at inhibiting of complex called LSD1.
This is work done by Doug Engel's lab
in which they treated sickle cell mice
with this RN-1 inhibitor
and saw increases in fetal hemoglobin
and reversion from sickling state
to a non-sick state.
And this is thought to be proceeding via mechanism
of PGC1-alpha increase
which leads to increases in fetal hemoglobin,
decreased sickling, and decreased hemolysis.
And interestingly, in terms of combinatorial therapy,
thinking about this, which I'll get to a little bit in my --
in some of my work, there's actually been work
coming that Don Lavelle worked on that looking at increases
in reticulocytes and gamma globin
when you combine decitabine,
as I showed you previously with LSD1 inhibition
via RN-1 showing additive or even synergistic effects
as far as fetal hemoglobin induction.
And this is also shown in non-human primates
in this study. So it'd very interesting
to see how this replicates in some of the --
in human and clinical studies that are ongoing right now.
And the final thing I wanted to mention
is a really interesting study
coming out of Bioverative and Betty Pace's group
looking at the role of Nrf2 activation
in fetal hemoglobin induction. So they had shown
previously that in treating with this compound,
dimethyl fumarate can increase Nrf2 levels
and that can increase gamma globin levels
and also increase heme scavenging and detoxification
and decrease reactive oxygen species.
And so, in relationship to this,
there's actually a study just came out this year
looking at the role of another compound
repurposing a drug that's already out there,
simvastatin, which is used for treatment of high cholesterol.
In which they showed,
at least in vitro that simvastatin treatment
could achieve high levels of fetal hemoglobin induction
and increase in stabilize Nrf2.
And so it'll be interesting to see if this holds true in,
in vitro models
and whether this is something that could be used potentially
in the treatment of sickle cell disease.
So I wanted to mention two stories
kind of that we're working on in lab,
one of which is a set of compounds called IMiDs.
So IMiDs are very interesting in that they recapitulate machinery
that's already present in cells. That's E3 ubiquitin ligase,
essentially a cereblon DDB1, CUL4,
and ROC1 complex that tags endogenous substrates
or degradation in the proteasome.
IMiDs actually bind to this cereblon pocket
and recruit neo substrates for degradation that's present.
And so this class of drugs was originally used in the 1950s
for morning sickness in women,
and was withdrawn because of it teratogenic effects.
However, in the 1990s IMiDs became used in the treatment
of multiple myeloma
a plasma cell disorder of the bone marrow,
and was approved both lenalidomide and pomalidomide
for treatment in multiple myeloma patients.
One of the things that's very interesting
that has not been recognized in the field as robustly,
is that IMiDs are actually very potent inducers
of fetal hemoglobin in vitro.
So originally work done by Griff Rogers back in 2007,
had shown that thalidomide at high doses
increases levels of gamma globin and F cell levels
and work done by Kyle Chan.
And Celgene actually showed this is also true
by in with lenalidomide and pomalidomide
at a significantly lower doses,
so much more potent induction of fetal hemoglobin
at much lower doses. One of the seminal works
and most comprehensive works that was done
was by Brian Del Mobitz and Leo Blanc's group,
in which they showed that pomalidomide treatment
decreases repressors such as BCL11A
and decreases other repressors such as SOX6 reverting cells
to a more fetal-like state
and acts early in erythroid differentiation,
unlike some of the other targets like HRI
that we've shown previously.
So the question is, is this also true in patients?
And so in this study,
they actually looked at multiple myeloma patients
that have been treated with pomalidomide
and asked after pomalidomide treatment,
do they see higher levels of fetal hemoglobin?
And in fact, they do in these three patients shown here.
This at the same time was a study,
a phase 1 study led by Abdullah Cutler
that looked at pomalidomide treatment
in sickle cell disease patients in escalating doses.
And remarkably, they actually saw higher levels
of fetal hemoglobin in this study
at the three milligram per day or higher dose,
which is similar to what's used in multiple myeloma patients.
So it's possible that, you know,
if we had a broader therapeutic window via other modulators,
could we increase levels of fetal hemoglobin
in a safe and effective way?
And some of the work we previously published
showed this may be true. So as I mentioned before,
HRI is this regulator of fetal hemoglobin.
If we co-deplete HRI and treat with pomalidomide,
we see significant levels of fetal hemoglobin induction.
Now, if we decrease the pomalidomide dose by tenfold
or a hundred-fold, we see a dose dependent decrease.
But if we treat and deplete this,
this dose by tenfold and co-deplete with HRI,
we actually see persistence of high levels of fetal hemoglobin
despite a tenfold decrease in pomalidomide,
which is interesting to me in that it's possible
we could maintain on-target effects
by limiting other off-target effects of drugs like IMiDs.
This is RNA-Seq data
that we generated indicating at high dose
we see high levels of gamma globin induction
and decrease in B-cell of an A.
If we decrease the dose of pomalidomide at tenfold,
we can see the same degree of F cell induction,
but half the number of genes changed.
So it's possible by doing, using combinatorial therapy,
we might be able to limit
some of the undesirable side effects of these inducers
while still maintaining high levels of our on-target effect,
which is increasing fetal hemoglobin
for the treatment of sickle cell disease.
And I think really, despite this really robust data
in vitro the clinical aspect of this,
bringing this to patients remains unrealized and I --
she just looked and it's very interesting,
this pomalidomide is being tested in a bleeding disorder
called hereditary hemorrhagic telangiectasia
and was just published eight hours ago
in the New England Journal,
that they're able to use pomalidomide
to actually significantly reduce bleeding risk in HHT patients.
The side effects people worry about with these drugs
are thrombosis mostly and neuropathy.
They did not see increased thrombosis or neuropathy
in these patients that were treated.
And so I think many of the side effects
we've seen in the past with IMiDs are more related
to the underlying clonal disorders and myeloma
rather than anything per se, to the drug itself.
So I think much more to comment, we're working on this in lab,
trying to understand the mechanism
and many of the others as well.
And bringing this to patients ultimately would be either
repurposing these drugs for treatment
or understanding the mechanism to develop better drugs
is a goal of the field too.
So I wanted to leave you with one last little story
and just a short story that's going on in our lab right now
involving a newly identified regulator of fetal hemoglobin
called PP6C or PPP6C.
So initially we worked a lot on HRI
and we actually are collaborating to develop
direct kinase inhibitors of HRI,
which is very exciting and have some really interesting data
in some animal models currently.
But we elected to perform a phosphatase knockout screen
to understand are there compensatory phosphatases
that might be regulating these pathways
and we understand better mechanisms
of red cell development and fetal hemoglobin control.
So we performed a CRISPR-Cas9-based screen,
again using HIC2-Cas9 cells and a phosphatase library.
And so ordered into fetal hemoglobin high in low cells.
And we can plot the data like this
in which we look at read counts on the x axis
and fetal hemoglobin low and a high on the Y axis.
Anything above the identity line indicates a gene
that when depleted leads
to higher levels of fetal hemoglobin,
which is what we we'd like to achieve.
And in five of the six guides tested,
we found a gene called PP6C or PPP6C.
So what is this?
So this is actually the catalytic subunit
and protein phosphate 6.
It's a holoenzyme complex that enables --
it's expressed in a variety of different tissues
and cell types.
But the specific regulation of some of these factors,
including three different PP6Rs,
and three different anchor and repeating domains
allows for very specific functions
in different cell types and tissues.
Previously, this has been described
in cell cycle progression,
innate immunity and MEK/ERK signaling pathways,
but has not previously been described
as a fetal hemoglobin regulator. And one of the intriguing parts
about this holoenzyme enzyme complex
is that one of the subunits, the PP6R1complex, is --
actually seems to be selectively expressed in red cells
at the time point at which we are making many of the fate
decisions involving fetal hemoglobin regulation.
So this could serve as a red cell selected target
for increasing fetal hemoglobin levels.
And I don't have time to show you all the data today,
able to show you a subset
in which we are able to validate these initial results
in primary human erythroid cells,
as well as in sickle CD34 erythroid cells,
which we deplete PP6C.
We see a doubling of F cells either at or
even sometimes exceeding BCL11A plus sgRNA depletion,
which is what's used in the clinical trials
that we just heard about in four different donors here,
and a significant increase in Fs,
in hbF by HPLC using both of these guides here.
We've also shown this to be true in xeno transplant models
and NBSGW and vivo mechanistically.
We think this proceeds, at least in part via BCL11A depletion,
we see loss of BCL11A
in a dose dependent manner following PPP6C loss.
And we've tested a variety of different
other targets downstream,
including many I've described here today
and we do not see any changes.
So we're still trying to understand
the mechanism connecting PP6C to BCL11A,
but we think this is the primary mechanism
that drives its effect on fetal hemoglobin.
And just to add you one last piece of data and far as PP6CR1,
we ask, can we deplete PP6R1directly
and recapitulate many of the effects
that we see by depleting the catalytic subunit?
So if we deplete either PP6C or PP6CR1,
we see a loss of both indicating they're both
required for stability of this hollow enzyme complex,
decrease or depletion of PP6C
or PP6R1 leads to increases in gamma globin
without changes in beta globin
and significant increases in F cells
that recapitulate the vast majority of the effects
we see with PP6C loss
indicating this could be a red cell selective modality
to be able to increase fetal hemoglobin
without off-target effects on other tissues and cell types.
So much more to come on this. We're actually in the process
of generating phosphoproteomics data
to understand what are the direct targets of PP6C
on either BCL11A directly or other intermediates
in the regulation of fetal hemoglobin.
But we think this could be a potentially interesting target
for modulating fetal hemoglobin
in sickle cell patients in the future.
Great. So I think I made it on time.
So I'd like to thank the organizers, Swee Lay, John,
Jennifer, and Monica.
It's really been a fantastic conference so far and much more
to come in the NHLBI for hosting the conference
as well as the NIDDK for funding.
My collaborators at UPenn, including the Blobel Lab,
the She Lab, Peslak Lab, and the Chondro's Lab
as well as our collaborators at Penn State
and my own lab that I just started this December
and have I be able to recruit a postdoc,
a couple of technicians and a biomathematicia
and very exciting work.
Hopefully more to come and many more meetings
where I can interact with really fantastic folks in the field.
So thank you. I'm happy to take any questions.
Haydar Frangoul: We have a couple online.
Scott Peslakak: Great.
Haydar Frangoul: We'll get the --
we'll get the questions here.
And there are a couple online. I want to leave time for them.
Swee Thein: So one, two of all of them.
One thing I may have missed, but I'm just wondering,
did you ever consider this PHI group of compounds?
There are now six of them that under clinical development
for treating an anemia in chronic sickle cell
chronic kidney disease. Scott Peslakak: Yeah.
Swee Thein: And in fact,
two or three of them were even been approved.
Scott Peslakak: Exactly, yeah.
Swee Thein: In Europe. And of course you --
for me, following the literature too, of [unintelligible]
actually increases fetal hemoglobin.
So I may have missed it,
but I just wonder what your thoughts
are on this group of compounds
Scott Peslakak: In terms of the,
the prolyl hydroxylase inhibitors?
Swee Thein: For repurposing them.
Scott Peslakak: Yeah, yeah.
Swee Thein: For treating sickle cell disease.
Scott Peslakak: Yeah. I mean, I think that's --
I think repurposing drugs
that are already approved for other reasons,
especially ones that don't impair levels
of hemoglobin and in fight --
in fact might increase hemoglobin
is beneficial, I think.
You know, I have talked with a couple of folks about this
in terms of we worry
about sometimes increasing levels of hemoglobin
and whether that's detrimental as far as viscosity.
I'm not sure that's as relevant
or as true if you have fetal hemoglobin,
how that actually contributes to viscosity.
So I think it might be useful,
even if it increases overall hemoglobin levels,
it could be beneficial.
I'm not as familiar with those --
with that specific set of papers,
but I think it's certainly repurposing drugs
to be able to treat sickle cell patients
in a way that we could test
in a more rapid cycle of clinical trials
would be beneficial, I think, for the field.
Male Speaker: So Scott, yeah, that was really terrific.
I have a question. Really just wondering
if you have any insight or inside knowledge
about the wiz degrader. So we were all like blown away
with the ASH presentation, right?
Yeah, but they -- but they showed only effort ticks.
Scott Peslakak: Yeah.
Male Speaker: In the non-human primate,
two of three had quite substantial rises
in effort ticks
[phonetic sp], but they didn't show fetal hemoglobin.
And I'm always very suspicious when
the last figure is effort ticks and there's no protein data.
And in between the time of the ASH presentation
and the publication, there's still no protein data.
Scott Peslakak: Yeah.
Male Speaker: So do you know whether they measured
and it was low?
I can't imagine that they didn't measure fetal hemoglobin.
So my assumption is that it's not high,
but it's a lot of cells with a little bit of fetal hemoglobin.
Scott Peslakak: Yeah. Male Speaker: Can you comment?
Scott Peslakak: It's a good question.
I can tell you that in the publication,
you know, they did see effects in most of their nine,
even primates, but not all of them.
And so that's an intriguing result as well.
You know, is there --
and I think this is an important question for the field,
are there signaling pathways that in certain patients,
patients we might modulate and we might not have effects?
And this is really important.
This is really why it's important to understand
these basic mechanisms,
because if we just develop lots and lots of drugs
and don't understand why, we have absolutely no idea
which patients are going to benefit,
for how long and in what combinations.
I do think combinatorial therapy is probably every other red --
every other blood disorder
thinks about combinatorial therapy.
And to think we can just have one drug
that's going to be the perfect drug for everybody,
I think is wishful thinking.
To get to your exact question, I don't know.
I do think that there --
I think the mechanisms that are underlying wiz
are probably different than those
with other cereblon-based modulators,
including pomalidomide.
But specifically in terms of the F protein level.
I'm not entirely sure. Yeah.
Haydar Frangoul: Okay.
There's a -- there are a couple there.
Yeah, go for it.
Manu Platt: Okay, so just a really quick question.
Sorry. Yeah.
This is also a great coverage of the field,
so not a clinician.
So you've kind of answered my -- your last bit, but --
so what's the idea that these other fetal hemoglobin modifiers
would be applied to patients
where either hydroxyurea doesn't work,
or like how do you see it rolling out to actual patients
and that insurance coverage would work
or that it would hit a new target
or that people's fear of hydroxyurea
would not be scared in these new modifiers?
How do you see that affecting the patients?
Scott Peslakak: Yeah, it's a really good question.
I will say there are scenarios where I worry about,
you know, generally hydroxyurea is very safe, very effective.
It is not shown to be toxic in any way.
There are scenarios where I worry
about using hydroxyurea for example,
after a bone marrow transplant or after gene therapy
where patients have gotten busulfan conditioning.
I think there is actually a higher risk
in giving hydroxyurea after that scenario.
I think in terms of whether that has been shown,
but that's what's been shown in the PV field
and previously actually.
I think in terms of being able to predict
and who might benefit and who might not,
I agree, this is a really incredibly important question.
Reduction remains a standard of care
for all patients with SS and SBE to zero disease
starting at very young ages.
And I think it's going to be a very long time
before that gets replaced by anything where we say,
no, we shouldn't give hydroxyurea.
But in patients, in adult patients,
we oftentimes have difficulty getting to maximum
tolerated dose because of neutropenia,
because of other side effects.
It's actually much more, I think in the pediatric setting,
we probably can push most, if not all patients
to get really good levels of fetal hemoglobin in adults.
It's much more challenging.
And so having other ways to modulate globin
switching other than Hydra in the adult phase of life,
I think is really critical.
And that's where we're going to see a lot of these be used,
I think.
Haydar Frangoul: Okay. There are a couple of questions online.
One was they want you to discuss the pros
and cons of the targets you mentioned
and which one is you are the most excited about.
Scott Peslakak: Yeah. I'm a little biased, I guess.
I can tell you that we're really excited about heme
regulated inhibitor and HRI kinase ambition.
I think the benefits of HRI,
this is red cell specific and HRI knockout mice
really don't show significant phenotypes
other than in severe iron deficiency
and maybe in thalassemia has been shown by Jane,
Jane and Chen's group.
But overall, HRI in inhibition is very well tolerated
and seems to be one of the earliest
and best targets that we know.
I think IMiDs are -- since are --
they're already approved,
understanding how these work and potentially
there are thalassemia physicians in Southeast Asia
that actually can't -- don't have access to blood
and need to increase levels of hemoglobin
and are using thalidomide off-label for patients
to be able to increase levels of hemoglobin
and, and fetal hemoglobin in particular.
So there's actually real world data.
This might be beneficial to --
not to sickle cell patients, but to thalassemia patients.
I think in terms of the molecules
that are out there right now I would be to --
Dr. Tisdale's question,
I'd be interested to see the protein level for whiz,
that's probably one of the more exciting ones
as far as effect size.
But I really think all of these compounds
have pluses and minuses,
and I do think it would be helpful
to be able to think about how these signaling pathways
are modulated at baseline to be able to choose better.
We're a ways away from doing that,
but I think Dr. Bauer's earlier talk about,
you know, actually thinking about how to predict,
you know, off target effects for his CRISPR guides.
We can do something similar.
I think with overall signaling pathways
in sickle cell disease patients, it's going to take a while,
but to do that, we need to understand how this works first.
Haydar Frangoul: The last question came in,
which I think is important, that nobody --
I mean, this is close to my heart, is SC patients.
You mean SC patients are a --
I mean, I can tell you that for the gene therapy trials we did,
none of them included SC patients.
And you know, anecdotally lock has,
it is when -- because Javi got approved,
the first three referrals to our site were SC
and we said no, because there is no data.
And I'm not aware of any data
in the persistent fetal hemoglobin world
to be convincingly showing that high F will help SC patients.
Scott Peslakak: Yeah, I think that's a great question.
I think part of the issue is that --
I remember speaking with Dr. Lydia Ecker
about this actually at a conference a few,
maybe about a few months ago, a year ago.
And really, SC is really an orphan disease.
It's -- we cluster it in with SS and SB to zero,
but I don't think we really understand
at a very basic level how SC works.
And why say hydroxyurea doesn't really work
in the majority of SC patients.
And I think -- so we now are --
I believe in the field, I haven't used this personally,
but we now are close to having a mouse model for SC
to be able to understand how this works mechanistically
and test some of these compounds.
I think you're right.
In terms of, we don't necessarily know
that F induce F induction improves SC,
but we think based on real world data that it probably does.
And I -- it's a vastly understudied,
but incredibly important field to study
because these patients have a tremendous
phenotypic variability. Some are as severe,
if not more so than SS disease and some are not.
And understanding how this works at a very basic level
is going to be critical to know what works best.
But I do think this could be beneficial many
of these compounds in SC patients as well.
Swee Thein: You know, I can agree with you more.
I think it's a very much understudy disease.
Everybody thinks a bit as another sickle cell syndrome.
It's complex.
I mean, even when the highest fetal hemoglobin I've seen,
I see is like 6 percent.
Even when you have the older hemoglobin at QTLs.
Whereas I've seen them as high as like 29, 30 percent,
not the Mendelian form of HF.
And then of course, there's this C component
that actually adds to the you know,
the viscosity problem and MCHC problem.
Yeah. So, and then of course, don't forget,
it can also co-inherit hyperglycemia.
That adds know even more of a interacting factor
in the disease differently to that hyperglycemia.
Haydar Frangoul: Well, thank you so much for the talk.
We are doing great on time. We are one minute early.
We have a break until 3:45.
Please reconvene at 3:45. Thank you.
Well, it is my pleasure to introduce our second speaker,
which is Dr. John Tisdale.
He is going to talk about the increased
anti-sickling potential genetic approaches.
Dr. Tisdale is a senior investigator
and chief of the cellular and molecular
therapeutic branch of the national Heart line
on Blend Institute, NHLBI, at the NIH.
It's my pleasure to let Dr. Tisdale take custodian.
John Tisdale: Okay.
Thank you very much for the opportunity to speak.
Thank you Jennifer and Monica for welcoming us back to Jamaica
and back to the Pegasus.
It's really an honor to be here again.
So I think much of what I'm going to say
has already been said,
but I can use some of the same slides
that you've already seen to make some different points.
This is a slide that I'm really happy to show here today.
This is a figure that my colleagues
Swee Lay Thein and Bill Eaton and I made just four years ago.
Okay. So we have these anti-sickling approaches
to sickle cell disease,
including current treatments like hydroxyurea,
reactivating fetal hemoglobin,
keeping cells from sickling before they get back
to the lungs, allogeneic transplantation
where we take somebody else's bone marrow stem cells,
do a transplant and fix the disease.
And then there was this dotted line
which separates those current treatments
from the future treatments.
So I get to move that line over now
because both gene addition and gene editing,
as you heard earlier have been now
FDA approved in December of last year.
So that's a big deal.
We finally have something that we can offer to patients
that's transformative, potentially curative,
that doesn't involve them finding a donor in the family.
And then I'll just update the results of the HGB-206 study
in the 210 extension, just in the interest of showing
this other anti-sickling approach.
And there'll be more information about this tomorrow,
both in terms of screening for genetic changes
that might influence results.
And also, you know, looking at patient outcomes.
But this is a study where instead of getting your cells
harvested and manufactured with CRISPR
cutting the BCL11A erythroid enhancer,
instead they go to a central manufacturing facility
where a lentiviral vector based on HIV
is used to transfer a copy of the beta globin gene
to those hematopoietic stem cells,
and hopefully in a high enough efficiency
that it provides for anti-sickling.
And in this case, there's a beta T87Q change
which is in hopes of making this more anti-sickling
than just standard beta globin.
So these have been published now groups
A and B in the first manuscript
on the left and group C on the right.
And I'll show you updated results that Julie Kanter
presented at ASH last year.
First, that there was a great hemoglobin response.
So we had this definition of hemoglobin response.
It was like, you know, an increase in greater
than three grams per deciliter of the total hemoglobin
or greater than 10 total grams per deciliter.
So there was a, so-called hemoglobin response,
and we wanted at least 30 percent
of that coming from vector.
So this is what we initially planned to be the,
you know, the outcome measure.
We wanted to see that the vector worked, it made hemoglobin --
it improved the total hemoglobin.
And -- but what we saw was that pain was also going away,
and that was a much more difficult measure.
Acute pain episodes that require hospitalization.
So-called severe VOEs.
The FDA wanted us to use as an -- as an outcome
because that's, you know, what was used for hydroxyurea,
for example. And for the globin response,
the majority of patients had this globin response 87 percent,
and all of those that reached it maintained it long-term.
And here you can just see the improvement in hemoglobin
over time in the fraction and blue coming from the vector,
very similar to what high dog
just showed for the CRISPR approach.
So these slides all look remarkably similar.
These are different patients, different approaches.
Again, to that same point, here's pain.
And this is severe vaso-occlusive events again,
as defined very similarly to the hydroxyurea study.
You see the red dots on the left, many of them.
Just a few red dots on the right,
most patients had resolution of the severe pain.
But there are dots remaining and that's, you know,
something maybe we can discuss. It keeps percolating up.
There are some patients with chronic pain
that require hospitalizations.
They were shorter when they came in,
much shorter than what they were afterwards.
But some patients are indeed still having pain.
And as was showed in the last talk,
there was near normalization of hemolysis markers.
So, you know, for some patients that normalize completely,
for some, they don't normalize completely as a group,
it's almost normal.
So this looks great, but there's still, you know,
maybe something going on there
that we might be able to do better
if we had higher efficiency of gene transfer
or a higher efficiency of beta globin expression,
or higher levels of fetal hemoglobin induction,
with the CRISPR approach. The, you know, the --
what we saw was typical of an auto transplant.
We didn't have any veno-occlusive disease
from the busulfan conditioning,
and there were no vector-related integration events.
And we had one death in group C from pre-existing --
what to be preexisting cardiopulmonary disease.
So the first patient has now been signed up.
The table here is responsible for that patient.
Maybe they can give us some updates.
I know that Gina Kolata wrote another article just yesterday
or something in the New York Times
because a patient got infused.
So there's another article just like this one
in the New York Times by Gina
cataloging the first patient here.
But we have new technology.
So this is a shout out for Swee Lay.
The reason for this slide.
You've already heard that this really important study
which demonstrated BCL11A as a quantitative trait
locus affecting F cell production,
really launched a whole new field.
And we and others wondered, you know, can we inhibit BCL11A,
cut the gene and cell for --
cut the erythroid enhancer once that was discovered as a way to,
you know, fix sickle cell disease?
And you've heard the whole story now unfold just before.
And just another shout out to Swee Lay,
she just awarded the Shaw prize,
which is, you know, like a Nobel Prize.
So [applause]
with Stu Orkin who did a lot of the work you know,
characterizing the erythroid enhancer
and all with Dan Bauer, as you heard earlier.
So it turns out we can do that.
Dan showed the bottom left panel earlier showing
that if you cut the erythroid enhancer,
you get just as much fetal hemoglobin
in the right red encircled bar
as you do by cutting the gene itself.
So this way you can make it erythroid-specific
and not affect anything else that BCL11A
might be doing in amato cord stem cells.
And in fact, we did some work with Dan,
where we tested this in the non-human primate.
And at first we did a competitive repopulation assay
where we competed BCL11A erythroid enhancer edited cells
with AAVS1 edited cells. So this should be inert.
So that served as our control, and we had equivalent in graft,
and we had no perturbations in hematopoiesis.
We had robust fetal hemoglobin.
And this looked like, you know, it could be a go in the clinic.
And in fact high note didn't show these data.
This is first New England Journal publication
with this trial.
And note here, this is a patient with thalassemia
where all of the hemoglobin is coming from fetal
and it goes to normal.
So there are no more transfusions
and a complete normalization of hemoglobin.
So it's working for thalassemia really quite well.
And he showed you this, you know,
just like this slide that I showed.
There are only a few dots after this approach.
And again, FDA approved.
So one thing that I did want to highlight is we've looked
at our allogeneic transplant experience.
Now, in 163 patients, it asks the same question,
what happens to pain?
Deepika had looked at one year,
and we saw in those patients at one year
that there was still quite a bit of pain
that patients were reporting and some pain
that required hospitalization.
But after two years in these 163 patients,
we saw almost no pain.
So in yellow is the first year, and in red is the second year.
And these patients are patients that rejected
where you would expect pain to return.
But there's still, you know,
a few patients who are having pain
even after successful transplant beyond the first year.
Well, HIV-based antiviral vectors have now,
you know, proven effective for a number of disorders.
You see here, you know,
there's ones for thalassemia sickle cell disease,
as I mentioned, adrenoleukodystrophy,
severe combined immunodeficiency.
So these vectors are being used in other settings
also successfully.
So we've made a lot of strides in the way that we do this.
You know, we can -- we can --
in graph cells that we've genetically modified
and prove that those genetic modifications
can reverse the disease.
But as has been mentioned several times today,
we have to use busulfan to get these cells
and it's really no fun.
We have to ablate the marrow, support the patient,
give transfusions of blood and platelets, give antibiotics
and then hopefully get the patient through.
So we've been looking to try to replace
that toxic chemotherapy with an antibody
that can be linked to a drug to see
if we can get engraftment at the same level.
And we've tried it with a couple of different antibodies.
This first one is to CD45,
which is expressed on all hematopoietic stem cells,
so we can target those cells.
We can use a site specific payload
to just rid the body of those cells and those cells only.
And in the case of CD45, we deplete the immune cells too.
So we could also use it in a --
in a bone marrow transplant for a brother or sister.
And for this, Dan already showed some of this.
We were looking at a double editing strategy
because if you edit at the plus 55
and plus 58 that he showed,
you can get higher levels of fetal hemoglobin
both in vitro here
and an engrafted immunodeficient mice when you edit it,
both plus 55 and plus 58 higher than either alone.
So we're looking at improving the efficiency
of fetal hemoglobin induction while looking
at whether we could get grafts in without busulfan.
And these are the monkey studies that we did with Bob Donahue,
who's here in the audience looking at double editing
for the plus 55 and the plus 58 and conditioning
with either busulfan or antibody.
And you can see the in vitro results
we had very nice fetal hemoglobin induction
higher with the double editing here up to 80 percent.
And when we grafted animals with busulfan shown in gray,
we got really nice levels of indels persisting over time.
These are weeks post-transplant. So these are many years out now.
So 80, 90 percent indels in the busulfan
treated group in the three animals that received antibody,
similar levels of indels
and similar levels of fetal hemoglobin induction.
What Dan didn't show is that later
we challenged these animals with phlebotomy
to sort of stress their erythropoiesis.
And you can see that they went right back up
to the levels that we saw right at the time of transplant
when they were indeed stressed.
And I think this is probably more like we would see
in the setting of thalassemia or sickle cell disease,
where, you know, they're having to work overtime to repopulate.
We also just marked the cells with the lentiviral vector
that had a barcode inside.
So we could see how many got in, how many got marked,
and how they contributed over time.
And whether it was similar to busulfan
because we wanted to answer the question
that was posed by Mark earlier, like you know,
do we get enough cells in with a transplant,
with this conditioning too have polyclonal hematopoiesis?
And indeed we did. We had a Shannon index,
which is a measure of the complexity of hematopoiesis
that was similar in the antibody treated animals
when compared to busulfan TBI or TBI.
And here you can see the contribution of those clones
over the first year in this animal in six months
and the second animal.
We then went on to look at antibody to see kit
which is more exclusively expressed on hematopoietic
stem and progenitor cells.
So CD117 in this antibody drug conjugate
we used in the context of a lentiviral vector
expressing a smear to BCL11A much like the children's --
the Boston Children's Study by David Williams.
And you can see we got equivalent vector copy number
in the antibody conditioned animals
when compared to the two busulfan conditioned animals.
We had similar levels of hemoglobin F induction.
But the one thing that was really remarkable
with the use of the antibody
is that the menstrual cycle did not change.
So it was one menstrual cycle per month in the females,
whereas they, you know, they were markedly reduced
in the busulfan and TBI animals.
So Bob had the brilliant idea of just sending these animals
back to group housing to see if they would reproduce.
And in fact, four of the six in the first mating season
had successful pregnancies.
So this is, you know, as good as it gets for mating in a --
in a -- in captivity four of six.
So this preserved fertility, which is a --
which is a big concern.
In fact, two of these animals were in the ICU together
before they went out, you know, to the island [laughs]
and re-found each other and reproduced.
So we also tried gene conversion
and for that we made the sickle mutation
and we were using HDR with CRISPR
because we wanted to see do these cells and graft well,
because this is another anti-sickling approach
that's really elegant.
You know, just make the beta S mutation back to beta.
That should work brilliantly, right?
But it turns out hemato partic stem cells are resistant to HDR,
and though we could get like 60 percent correction,
which was making the beta S mutation in those cells,
they didn't even graft well.
So up top you can see they dropped
to like 1 percent in these two animals, HDR
but the indels persisted, you know, 40 and 20 percent.
So we already knew we could make indels
and they engraft and they'll persist.
But if the cell has to go all the way through homology
directed repair, those cells either don't engraft
or, you know, we're not really getting into
the amato cord stem cell with HDR.
So that looked like a strategy
that maybe we should leave aside.
And I think Graphite Bio learned this lesson the hard way
in their first patient where they saw similar levels.
So now Hena, but in the lab, is looking at strategies
for comparing anti-sickling approaches for sickle cell.
So she's looking at gene edition, you know,
with the BB305 vector BCL11A disruption
with CRISPR-Cas9, and base editing,
which we've chosen to make them a Cas mutation.
This is a -- this is a hemoglobin variant
that should be -- should be benign.
And then we can look to see how well we do
whether we inhibit sickling equivalently
and then put those through the animal models.
So, so far she's shown that she can get really good editing.
So, you know, with the BCL11A editing,
we've gotten really good at that.
We get 95 percent pretty much every time.
Makassar editing, not bad, about 80 percent.
Not as good as BCL11A, but pretty good.
Vector copy number for the T87Q vector of round one.
So all of these are what, you know,
what you would get typically in the clinic.
And fetal hemoglobin induction with the BCL11A
editing of 60 percent,
40 percent endogenous with the T87Q vector.
And then when we look at anti sickling in Wan Lee
and Bill Eaton's assay which has been very predictive for us
even in the clinical setting,
we can see a really nice inhibition of sickling
and really almost no sickling frequency in these cells
after differentiation into red cells ex vivo.
So it looks like when we do well
and we have an efficient strategy,
that the anti-sickling properties are pretty similar.
And she's now comparing them in the mouse and in fact,
harvesting them now for secondary transplants.
And for a look at the hemoglobins,
because you can only really get red cells out of the marrow.
So we do this at their four-month time point.
But then, you know, there's all this use of this mouse model,
this immunodeficient mouse model to teach us
whether strategies are working.
And I've always been sort of reluctant to accept those
because when I first started in the field,
we used mouse data to justify
our human transplant marking studies.
And, you know, we could get 50 percent in the mice
and we got like one in a million in the human.
So there was a big disconnect.
But these transplants where you take human cells
and you put them into immunodeficient mice
may be better than just transplanting mouse into mouse.
So we've been trying for years
to make this rhesusized mouse model.
That is, we take rhesus CD34 cells, not human,
and put them into the mouse
because that way we could do parallel studies
when we do a transplant in the monkey
and we get 10 percent of whatever,
if we took a fraction of those cells and put in those mice,
and we also got 10 percent. We would be much more likely
to believe that that mouse is a relevant model.
And we've just never been able to do it,
because every time we've tried it doesn't work.
So Hasan Demirci in the lab decided to try again.
And this time it worked.
We got really good engraftment in these mice
actually higher with rhesus cells in blue here.
The reason it falls off later is because it did so well
that the mice got graft versus host disease at the six months.
So we started losing animals except for the ones
that were only engrafted at low levels.
But suffice it to say, we now have robust engraftment.
We can look to see whether we can predict with this model.
And one of the things that we did is we took animals
that we've had for years,
like these two animals that we've had, you know,
for years with marking in their blood
with green fluorescence protein or yellow fluorescence protein
when we were doing competitive repopulation,
looking at different assay,
I mean, looking at different vector designs.
And in this animal we had almost equivalent GFP/YFP.
So 2 percent, 5 percent.
And you can see the readout in this mouse is,
you know, around 5 percent.
So very similar to what the animal has been showing us
for now many years.
And in this animal where there's a much bigger difference
between GFP and YFP 15
and four, we see, you know, around 25 and five.
So it seems to be recapitulating at least what we have in vivo.
So we're hopeful that this will help us to to move forward
in assessing these approaches.
But one thing that also keeps bubbling up here
is that this is hard to do,
you know, even in -- you know, in well-resourced countries.
I'm not really sure what to call it.
Now, knowing that this line
is so low in terms of income for country.
But you -- we really need to be able to do this
in a different way than a transplant.
So Francis, who's already been alluded to by Wilbur earlier
was giving a talk at ASGCT a few years ago,
and he really said it right.
You know, we have to come up with a strategy
because even before acknowledging that, you know,
patients are spread across Africa and India, we just --
we don't have the ability to do this.
It's just out of the question.
We don't have the ability to do it.
So we need to make some in vivo gene editing strategies.
So we're working on that now so we can get rid of the need
for all this, you know,
harvesting, processing, gene addition,
gene editing, freezing, transporting,
you know conditioning to where we could make it --
you know, we could take these tools
and stick them in a viral vector or nanoparticle and just inject
and hopefully have that go in and fix the patient.
That way we could just,
you know, make it work all in one visit.
And so we've been looking at antibody drug conjugates,
as I showed you, for depleting of amato cord stem cells.
But that proved to us that we can reach these stem cells.
So this CD117 targeted ADC,
goes in and knocks out the hematopoietic
stem cells equivalently to busulfan.
So if we could take this antibody
and hook it to a vector or hook it
to a lipid nanoparticle, maybe we could get in.
So even looking to make lentiviral vectors
and, you know, to put on their surface these antibodies.
We've done that with a C kit antibody
and in a cell line that variably expresses C kit,
you can see after screening by phase display,
a bunch of different scFVs, we found a number of them
that are very selective for CD117,
positive cells over CD117 negative cells.
And we're incorporating those into vectors now
to try to get a transfer.
And we can also do the same thing with peptides.
So we're using peptides
to basically decorate lipid nanoparticles
because it's a lot easier chemically to get a peptide
hooked to a lipid nanoparticle.
And we've, you know, screened again with phase display,
this time both with negative selection.
So we start them first on the negative cell line
and then on a positive cell line,
and then sometimes rescreen and then select the media
and the cell lysate. So we know cells that are --
you know, peptides that are binding,
and then peptides that are binding and internalizing.
And these are some of the results we get.
We've done it with a bunch of different levels of plasma
just because we thought that might interfere
and we find overlap between external and internal.
And then we start looking to see if those can be used.
And these are just some of them that, again, we've screened
for transfer of GFP in this positive negative cell line.
And again, some that are very selective for the CD117
positive cell line.
So I'll summarize by saying that, you know,
gene therapy works.
Finally, the last time I was here,
we were, you know, hoping to be here.
I think with improvements in the anti-sickling properties,
we should be able to make it better.
Of course, we need long-term follow-up.
Toxicities are still a problem. Hopefully we can get antibodies
working so we can get away from this toxic chemotherapy.
And of course, you know, if we could figure out a way
to deliver these genetic tools in vivo,
we should be able to reach where the patients mostly are.
And I think, you know, delivery through antibody
or peptide methods could help us do that.
And, you know, global distribution of vaccines
that were based on lipid nanoparticles,
the same lipid nanoparticles
that we're actually using in these --
in these studies were used for the COVID vaccine
that was distributed widely.
So we think we have at least infrastructure available too,
to roll that out if, you know, we can make it work efficiently.
So I'll stop there and acknowledge all of the people
that have helped and take any questions.
Haydar Frangoul: I'm going to start with my first question.
So you have a lot of experience at the NIH using,
you know, haploidentical transplant
with patients that having mixed chimerism.
You know, in gene therapy,
those products we are infusing into patients
are not a hundred percent edited.
So some products are 60, 70 percent edited.
In your patients who are mixed chimerism, long-term post haplo,
do their hemolysis markers totally normalized?
John Tisdale: No.
So I actually have a fellow who's working on that now.
Actually she's faculty now at, at Children's.
And she's also observed that patients
with mixed chimerism also have --
as at -- you know, as we've seen in all these patients,
mild elevation in reticulocytes as compared to those
who have full donor chimerism in all compartments
where the reticulocyte count seems to be normalized.
And if we look by flow at those reticulocytes,
we enrich for hemoglobin S.
Haydar Frangoul: Interesting. Okay.
John Tisdale: Mark.
Mark Walters: Hey. Hey John. That was a great talk.
John Tisdale: Thanks Mark.
Mark Walters: I wanted to pick your brain
a little bit about HDR in the hematopoietic,
the true hematopoietic stem cell
because I we've reviewed both Cindy Dunbar's paper
about that topic in the non-human primates
and then the data you presented today.
Have you tried ways of optimizing the health
of the hematopoietic stem cell through cycling
and media manipulations or inhibitors of non-hemology
and joining as a way to improve that outcome?
And or do you have other ideas about
how we might accomplish that in the future?
John Tisdale: So, yeah. So these two animals,
we had optimized all the way up to using,
you know, multiple adjuvants in the --
in the cytokine cocktail that the cells were cultured
in along with I53, I53 to inhibit 53PP1.
So that's inclusive of those, what what I've shown.
We've tried to further optimize
and we haven't been able to make it
any better than what we've seen so far.
We've been sort of banging our heads against the wall.
And so, you know, we've tried base editing, prime editing,
and those seem to be working now
with the same efficiency that we can get.
We can get 60 percent HDR with CRISPR,
but we can also get 60, 80 percent with prime editing.
And we can get hemoglobin CESAR
as I showed at about 80 percent with base editing.
So we think there may be advantages
to just nicking the DNA
as opposed to double strand breaks
that you get with these newer editing methods
that we're now pursuing. So --
Mark Walters: Yeah. Great. Great talk John, as always.
Do you -- to follow-up on Dr. Walter's question,
do you think that if we're able to get that level of HDR
or prime editing or anything else,
do you think it'll matter what the age of the stem cell is?
And do you think if --
say older patients where we try to do this,
it might be more challenging to get efficient editing
or about the same?
John Tisdale: Yeah, I do think age
is going to play a role both in the efficiency of editing
and, you know, in the engraftment long-term
and the dangers that might befall the patient having,
you know, started at an older age
and then have their hematopoietic compartment
whittled down to a much smaller number to reconstitute.
So I mean, I think it's true for most of the things
that we've tried to do in gene therapy
is that younger is seems to always be better.
Mark Walters: Yeah. Okay. Thanks.
Haydar Frangoul: Okay. Well, thank you, John.
It is my distinct honor to present the next speaker.
A lot of the work that I talked about,
a lot of it has to do with the work you did.
So again, congratulations on the big award.
Dr. Swee Lay Thein is going to present
increasing anti-sickling
potential pharmacologic approaches.
She is a senior investigator and chief of Sickle Cell branch
within the National Heart, Lung and Blood
Institute of the National Institute of Health.
Thank you.
[applause]
Swee Thein: Thank you, Haydar. It's a pleasure to be here.
So let's see. Do I just -- okay.
So I'm going to talk about anti-sickling approaches
using pharmacological means.
Oops, what did I do now? I've broken this. Okay.
I've nothing to disclose,
but I will discuss some off-label drugs.
So this is the outline of my talk.
First, I'm going to give you an overview
of sickle cell pathophysiology, just a very quick one
because I know all of you're very familiar with it already.
And the developments in therapeutic targets.
I'm going to talk about
the current pharmacological antidisciplinary approaches
and the scientific rationale and the possible concerns
that have been raised on compromising oxygen delivery,
utilizing approaches based on increasing
hemoglobin oxygen affinity.
And then when time permits also talk about
other potential pharmacological anti-sickling approaches.
So just to remind us about the Global Burden
of sickle cell disease,
and we've heard a lot about it today,
already, currently is estimated that there are 7 to 8 million
sickle cell patients worldwide and migrations actually changing
the sickle cell disease landscape.
This is a very simple representation
of the pathophysiology of sickle cell disease,
which we know is caused by the presence of hemoglobin S
due to a single base mutation.
And this abnormal hemoglobin S polymerizes when oxygenated,
and it is the root cause that leads to the change
in the red cell structure and function,
what we refer to as sickling.
So here the current therapeutic options.
We've all heard about transplant genetic therapy.
The four drug approved options covering the hydroxyurea,
voxelotor, L-glutamine and crizanlizumab.
Each of these have their own limitations.
And at this point, I'd also like to mention that crizanlizumab
has been withdrawn at EMA in Europe and the MHRA in UK
has revoked approval in 2023 for its lack of efficacy.
And what is recalled treatment in Europe in January, 2024.
So I want to talk about hydroxyurea.
Clearly then, there is an unmet need
for an effective disease-modifying drug
that can be taken orally.
We're all working towards getting more treatment,
some in qualified curative therapies,
but a large majority do not.
Especially the patients that I look after
who are adult patients
who becoming more sensitive to hydroxyurea
and they need something actually to improve the quality of lives.
So hemoglobin S polymerization, as you all know,
is the root cause of sickle cell disease
and it polymerizes only when it's oxygenated.
Increasing oxygen affinity
to inhibit hemoglobin S polymerization
has been discussed for years even by Ernest Butler.
And the tremor itself is --
you know, it moves from the low oxygen affinity state,
which is a T form to R. You know, very rapidly.
And the idea then is if you can keep the --
it in the oxygen-bound form,
then this will actually inhibit hemoglobin as polymerization.
So voxelotor or oxybryta or GBT440
was FDA approved conditionally in November,
2019 for treating anemia and sickle cell disease
in people were 12 years and older.
And actually, initially, it was started for treating.
You know, the endpoint was VOCs,
but they noticed that electro hemoglobin increase,
so then it would change then
to an endpoint of hemoglobin increase.
So it is a small molecule that stabilizes the hemoglobin
in an oxygenated state
to reversible binding to the amino-terminus
of the alpha chain of hemoglobin.
And this potentially inhibits hemoglobin S polymerization.
So there have been many studies now,
Phase I/II, Phase IIa, Phase III.
All you know, randomized double blind placebo control
and then we show to generally say, well tolerated.
And it -- patients show an increase in hemoglobin with a --
accompanied by reduction in hemolysis
and also sickling parameters,
which is consistent with this effect in reducing sickling.
And mostly, recently, there's a prospect study,
which is a post-marketing open label observation study
where they followed 150 patients
and they've been followed for five years
and they confirm the improved hemoglobin
and reduced hemolytic markers.
So the principle of increasing oxygen affinity
to inhibit hemoglobin S polymerization
is quite straightforward.
Because when hemoglobin contains oxygen and offloading,
it reverses to the T state, when it's de-oxygenated.
And that's when it polymerizes.
The problem though is as you can see, that when you --
a drug binds to -- in the oxygenized state,
the binding has been released quickly when oxygen is needed.
But actually in voxelotor case it is bound very tightly.
And what I'd like to show you this is.
Oh, sorry. How do I go back? So here is actually --
well, you can see the panel on the right
is a real patient that we treated and the blue line
is before the patient received treatment.
And the gray line is what we actually saw
in the oxygen association curve. The dotted line is expected.
And here we calculated
that there was 21 percent modification.
And you can see there's a little bump
because that's a bit that has moved to the left
when it is oxygen-bound.
And when it is treated with 200 micromolar,
is fully saturated, it's completely shifted to the right.
So as I said, there were concerns that, you know, okay,
overall it is safe -- it's -- there was no reduction in VOC
but there was no increase in VOC either.
But there was significant increase in hemoglobin
with reduction in hemolytic markers
and improvement in the surrogate markers.
The question then is, will there be compromised oxygen delivery
in the long-term?
And this was a concern that was raised by Bob Hebbel
and Hadland very early on in 2018.
And he's wrote this thing about rising hemoglobin.
It is a pyrrhic victory.
I had look up this word because I didn't know what it means.
And it's actually --
it means that it is one that is not worth winning
because so much is a loss to achieve it.
Because you know,
you have 30 percent modifications of the tetramer
and this raised the hemoglobin of course
because it used to reduce sickling.
But if it's 30 percent,
then you have to improve the hemoglobin 8 to 11.4
simply to acquire the 30 percent of oxygen delivery capacity
that is now lost by binding the drug in the first phase.
So that was a concern. And then, you know, Bill --
I worked with Bill Eaton and we were also worried.
So he carried out in vivo modeling
or actually vivo modeling red cells,
oxygen disassociation, sickling, and also just didn't look
at how they, you know, deliver the oxygen.
So what -- if showed that, in fact, voxelotor really --
it monthly reduces sickling when you do the sickling curves,
but it only actually let go of his oxygen
at very low oxygen pressure, like 2 percent.
And this is a situation where we see in the kidneys.
And it was accompanied by a very nicely written commentary
by Charles Quinn and Russell Ware.
And they in fact wrote this commentary
saying there's a very fine delicate balance
between oxygen affinity of hemoglobin and oxygen delivery.
So increasing oxygen affinity should delay the polymerization,
but at a potential cost of decreasing oxygen delivery.
And essentially, in sickle cell disease,
to have and to hold may not always be the best approach
[laughs].
And -- but we need perspective study,
longitudinal studies to see how it affects tissue perfusion
like NIRs and the brain MRIs. And I believe that there are
current studies undergoing for that.
That means more studies reported,
but it's very difficult because they're short term
and there's only a very small number of patients.
So the next trial, which shows a lot of promise
but it's not FDA-approved yet, is activating pyruvate kinase.
And this is anti-sickling.
And the clues to this actually have been laid long ago even.
First, I was just saying to [unintelligible]
last night --
you know, we referred a patient to sickle cell trait,
but she had severe sickle cell disease.
The only thing I could find in --
find in her was that she was PK deficient
and she's inherited one mutation in PKLR.
And in fact, when I looked up the literature,
somebody else in France,
Michel Cohen-Solal reported another case in 1998.
And in his patient he could measure the DPG levels.
They were very high and he showed
that it affected the oxygen affinity with very --
and then very high 2,3-DPG.
2,3-DPG is a substrate in the glycolytic pathway
and it is a key player in oxygen dissociation.
The other thing was noted is that Sam Charache
already observed like 50 years ago,
more than 50 years ago, that 2,3-DPG is very high
in rich cells bringing sickle cell disease.
And this was confirmed
by a more recent publication in Nature Medicine.
And finally this clever --
this idea that we could perhaps reduce
due to DPG in sickle erythrocytes as a modality
for ameliorating all the complication sickle cell disease
is really not new.
It was actually proposed by Poillon in Blood 1995.
So, it took us such a long time,
but because we needed a PK activator to come along,
just to remind you of your -- okay, the two key factors
that promote sickling sickle cell disease.
2,3-DPG is very high in patients with sickle cell disease
and increasing 2,3-DPG decreases oxygen affinity.
And as you know, hemoglobin S polymerizes
only when deoxygenated
and it stabilizes the S in the polymerizing form.
We also know the ATPs decrease in sickle cell disease
and ATP is very important in maintaining the hydration
and maintaining the iron and water hemostasis in red cells.
And we have -- decrease ATP,
the increased red cell rehydration,
increased hemoglobin S polymerization,
and all the problems that come with sickling.
This is just a summary
of what are the ongoing clinical studies suffices to say
that we completed the first Phase I dose escalation study
showing that was generally safe.
And now we have an extended study
and there's an open label study
in the Netherlands it's undergoing.
So Agios has now moved on and take --
started the Phase II/II
randomized placebo control study.
Phase II completed the enrollment and now in fact
Phase III, which is multicenter,
I think 101 hundred centers globally is already closed
because it's multicenter
and likely to have the main complete enrollment in October.
Now there's another generation also of PK activator,
which is manufactured by Agios and it's called AG-946.
And this has also been shown to be generally saved
and increased ATP decreased DPG
in keeping with the mechanism of the drug.
And another company which is -- which was former therapeutics
now brought over by Novo Nordisk is etavopivat or FT-4202 then.
And they also published recently Phase II/III study
showing that it was also safe and led to hemoglobin increase,
decreased DPG, increased ATP, all promising favorable results.
So this is not to say that you know,
there are no concerns about PK activators.
What do we know so far on the mechanistic implications
for clinical benefits?
Metaevent and etavopivat activates the endogenous world
type PK
and generally living shouldn't be safe and tolerable.
It increases ATP, decrease DPG levels
in a dose-dependent manner in keeping with its mechanism.
It improved hemoglobin, reduced the hemolytic markers,
increased oxygen affinity and reduced sickling.
There were no increase in VOC. In some small studies suggestion
that there was a decreased VOC frequency,
but I think this is still relatively short-term studies
and a small number of patients.
The same concerns that were raised was raised for --
voxelotor was also raised for these PK activators.
Will decrease due to DBG levels,
compromise oxygen delivery in the long-term.
And in that same commentary by Bob Hebbel,
he said while decreasing
DBG actually instigate oxygen offloading but --
and it doesn't involve any changes in the --
oxygen hemoglobin conversion
is not without physiological impact.
And the more recent editorial by Frank Bunn,
also cautions us to the --
you know, that we have to evaluate properly new drugs
which are designed to treat specific anemia
that should include a thorough understanding
of the underlying pathophysiology
with a focus on enhancing oxygen transport.
So what is the difference
between these antidisciplinary mechanisms of voxelotor
and PK activators?
I think it's very relevant to understand that.
Well, enhancing PK activity -- okay, decreases 2,3-DPG
but it also increases ATP.
So decrease 2,3-DPG, destabilize hemoglobin S fibers.
And this oxygen dissociation and binding of 2,3-DPG
to hemoglobin S is rapid unlike that of voxelotor
which actually binds very tightly
until very low oxygen pressure, like, 2 percent.
Another thing is that when you decrease 2,3-DPG,
it is also accompanied by increased pH
and a decreased MCHC which are also anti-sickling.
But I think as I come to kind of work with mitapivat,
it's the increase in ATP
which is actually the main factor that --
for the clinical benefit.
Because ATP is essential for RBC hydration
and the membrane integrity.
Another thing is that we in --
under trying to work out the mechanism of the mitapivat,
we found that it actually reduces
the tyrosine phosphorylation of band 3,
which is a key player in maintaining the red cell
membrane skeleton integrity.
There were other concerns that were also raised
about activating PK in sickle cell disease,
which was raised by none other than Jane Little, you know,
[laughs] who says, you know, PK activators could actually --
because by improving the lower glycolytic flux,
which is the one, you know,
below the hexose monophosphate shunt,
we could actually affect the HMP shunt,
which is the primary source for recycling NADP.
And this could compromise the ability of the red cells
to combat oxidative stress.
But in fact when you activate PK,
it actually increases the whole glycolytic flux
and there's increased glucose uptake.
So I don't think it's too much of a concern to worry about.
And I mean knowing beta-thalassemia mouse,
mitapivat actually increase the GSH and GSSG ratio,
suggesting that this HMP shunt
is actually increased by mitapivet.
More recently, we then also carried out
a multi-omics analysis of red cells in patients
with sickle cell disease on our extended treatment.
And on the left here, you show --
I show you the heat map of the mitochondrial proteins.
And what was so remarkable
was that within two weeks of taking mitapivat,
the mitochondrial proteins, in fact,
dropped tremendously to -- in -- consistent with the --
with the increase in hemoglobin.
And this is accompanied by a very nice commentary
from Andrea Glenthoj
and would show that actually increasing --
activating PK, actually goes beyond the ATP increase.
And it has a lot of multiple beneficial tropic effects.
So another concern that was raised
is when you withdraw hemolysis
and mitapivat abruptly discontinues.
I think this has been reported in the patient's PK deficiency
and it appears to be patient dependent.
In our experience, I've had two patients who abruptly stopped.
One left his medicine bag
which contains hydroxyurea and mitapivat in the car
when he was rushing to catch a plane.
He was without it for 10 days
and actually we were worried sick.
We tried to -- in the --
sending by FedEx to where he was saying,
but he didn't appear to receive it.
When he came back his hemoglobin dropped,
he was very tired and exhausted but he didn't get a VOC.
So of course I think this is patient-dependent
but nonetheless all patients should be fully informed
of the risk and medication adherence reinforced.
So I want to come back to the band 3 tyrosine phosphorylation.
Because this is a clue that has now led me
into the next potential anti-sickling treatment.
One thing which we notice in our dose escalations study was that,
why did the hemoglobin increase,
which we notice persists even after we stopped mitapivat.
You know, that we stopped it for four weeks
and it remained at the same level.
The only thing I could think of
is there's increased red cell survival
and the key culprit is such a band 3,
which is an intermembrane protein.
It has two main functions.
It's anion exchange protein responsible
for the oxygen and carbon dioxide exchange.
And it's also crucial for maintaining its integrity.
So what you see down here is cartooned.
The band 3 is the N-terminal, you'll get two tyrosines there.
They are responsible for actually interacting
with the anchoring spectrum skeletal complex
that maintains the integrity of the red cells.
And when it is fossil-related, it actually dissociates
or interferes with the association.
And these two sites, actually docking sites for S kinase.
What we found was that mitapivat
actually reduces this band 3 tyrosine phosphorylation.
So what I'd like to show you is you look at the bottom panel,
which is in blue
and that is a band 3 thyrosine phosphorylation.
And it shows the dose-dependent decrease.
On the top you see that this increased binding with PTP1B,
which is actually the protein tyrosine phosphatase 1B,
which is the -- constitutes phosphatase.
And there's also increased binding with anchoring.
And we know that it is not true inhibiting sick
because of the increased PTP1B.
Because the phosphorylation of band 3
is actually a balance between the kinase activity
and it constitutes phosphatase activity.
So mitapivat, which we think through ATG,
increases the calcium pump to move out.
So there's more PTPB coming in.
Affects it by reducing the band 3 tyrosine phosphorylation
that way.
So then the idea either is tyrosine phosphorylation
band 3 impacts integrity risk of membrane.
So if you can reduce the tyrosine phosphorylation band 3,
this has a potential as a sickle cell disease
modifying therapeutic agent.
And I love this diagram of Annu, plus in his review.
Because now I really think sickle cell disease,
we should think of it as a biomechanical disease.
The hemoglobin S polymers may be the root cause,
but it is those damaged red cells
which are the rascal that carry all the damage.
So this -- on the left side here
is the vicious cycle of sickling.
So if you can come up --
somehow interfere with the shape change,
and I think if we can inhibit SYK or spleen tyrosine kinase,
this will reduce the tyrosine phosphorylation
restoring its function,
restores the red cell membrane integrity,
will reduce fragmentation.
The red cell membrane, blebbing, reduce the micro-vesicles
and all the other erythrocystic DAMPs
which trigger the inflammation.
So S inhibitors can break this vicious cycle.
And so with that in mind we look at those SYK inhibitors
that have already been FDA approved
for treating other diseases.
Of course there's matinib,
but fostamatinib was kind of at hand.
So we tested fostamatinib
and his active biology compound is R406.
So what you could see is that Fosta actually showed
a dose-dependent --
the band 3 tyrosine phosphorylation reduce
but this dose-dependent decrease with the dose
and also with the same dose increase in time.
And on the right here --
side here is the loci to show it's deformability.
So on the top is -- you see the hemoglobin AA cells.
It didn't affect AA cells at all
because AA cells hardly have any band 3 tyrosine phosphorylation.
The band 3 is not so heavily in tyrosine phosphorylation.
The round circles are the SS.
And here you could see the blank line is a control
and there's a dose-dependent increase or decrease --
increase in the formability,
which is measured by an elongation index,
meaning that the cells can be stretched better.
And the bottom is using R406, the active biological compound.
I'm almost done. Time's up. Okay.
You can hang me off if you want [laughs].
So this is the sickling curves which I did with --
worked with Bill Eaton.
And essentially for those of you --
of you who not familiar with this assay,
they're subjected to slow the oxygenation and we --
the parameter we use is that what is a fraction of the risk,
the cells that are sickle at the -- no --
how long does it take for 50 percent of the cells
to be sickle?
So the longer the time the better.
And here we saw R406 and fostamatinib
and these curves are very similar to mitapivat
when I first started the essay.
So just to sum up now, we -- I spoke about mitapivat,
voxelotor, but this -- don't forget
reducing intracellular hemoglobin concentration
is also one key mechanism and under development
is restricting iron and erythropoiesis.
It's not practical, you know,
for patients who reduce iron intake
but is a clinical trial investigating
the utility of ferroportin inhibitor,
which is when vamifeport.
But I would like to add a sixth one to Bill's list.
Bill's list that maintaining integrity of red cell membrane
and the skeleton integrity is very important.
So I just -- I just want to acknowledge
and thanks on key collaborators, and thank you very much.
[applause]
Haydar Frangoul: Okay. We'll take some questions
and I have one online question if there is none.
I can ask the online question
while they are finding a microphone.
Based on your understanding of the mechanism of PK activation,
to what extent do you think mitapivat
that will reduce rate of pain crisis
in ongoing Phase III trial?
Swee Thein: Well, in the Phase II
which is standard review,
there are already some suggestions
that it reduces pain frequency.
But this observation is only for 12 weeks.
Haydar Frangoul: Okay.
Swee Thein: And for those of us
who looked after sickle cell operations,
they do have up and down periods.
We need to assess it for a longer time.
I think Jane has a --
Female Speaker: Yeah, I had a quick question.
And I have to clarify.
Actually, Victor GoDuket [phonetic sp]
is the one who pointed out the possible risk to me
in a conversation with another person.
And I don't want to take --
I mean I thought it was really smart of him
to ask that question about the --
you know, whether the oxidant stress would be more --
Swee Thein: Please.
Female Speaker: Sorry. But I guess my question
is an add-on to the one you just had,
which is for all of these agents that improve hemoglobin,
I mean that is the central problem,
but the pain from the BMT stuff sounds like it takes months
to maybe a year to go away once the red cells are fixed.
How, where do you think these -- you know,
PK activators and oxygen affinity
changer will land in resolving pain?
How long do you think that'll take
if you use the bone marrow transplant
as kind of a model of that? Or do you think that's relevant?
Swee Thein: Well, I think there's open --
but you know, I'm thinking about the patients that I look after
who don't meet that -- your eligibility criteria,
for care eligibility or transplant
and will become very sensitive in hydroxyurea.
And at least hydroxyurea is -- increased the hemoglobin, right?
Meaning they increase the hemoglobin.
But they still get sickly pain.
But whatever it is in hemoglobin, they can make --
will try to maintain the integrity and the cell survival.
So at least they can have a good quality of life.
And I do have patients here who have done extremely well.
Female Speaker: I guess, I'm just asking if you have a --
if you break a red cell and it gets inflamed,
you know, you get the white cells inflamed
and the endothelium inflamed
and then you stop breaking the red cells.
How many weeks or months do you think it'll --
or do you -- it'll take before that process breaks?
Swee Thein: Well, I think if you can break it early --
in fact I was showing the vicious cycle
there that you know, the pain that comes in an acute attack,
it is actually from the -- to me I think it is --
a lot of is actually in the red cell.
When it breaks, it releases the, hemo,
it releases the self mitochondrial DNA
and these are the inflammatory triggers.
And if you can actually contain it
then we can stop it blowing up.
Female Speaker: Thank you.
Haydar Frangoul: Scott, do you still have a question?
Scott Peslak: Yeah.
Haydar Frangoul: Time for one more quick question.
I will do a last speaker.
Scott Peslak: Great, great talk, Swee Thein.
I -- you know, to come back to the SC conversation,
I was just curious about what your thoughts are.
You know, the mechanism you're saying
is it can improve hydration of the cell, right?
And that's one of the things that we see in SC.
And so I'm curious the population that I have.
The biggest challenge with our patients
with high hemoglobin and SC disease
where we worry about driving hemoglobin
too high with drugs like mitapivat.
And I'm curious what your thoughts are
and what the risk is for that in those higher hemoglobin patients
as to whether it's offset or if there's any risk there
for driving hemoglobin too high.
Swee Thein: I haven't treated any SC patients yet in my --
it's still under trial, of course.
And if it is more ATP that is increased,
that is the, I think,
the main benefit underlying the mechanism of it improvement,
I think it would actually should help see patients.
And I believe it is the same mechanism
that, you know, underlies the benefit in PKD patients
and in thalassemia patients.
Haydar Frangoul: Well, thank you so much.
Our last speaker is Dr. Edward Benz.
He is presenting the emergence of genetic therapies
for sickle cell disease,
a curative treatment or treatment with curative intent.
Dr. Benz is president and CEO emeritus
of the Dana-Farber Cancer Institute and Director Emeritus
and principal investigator
of the Dana-Farber Harvard Cancer Center.
Can you hear us? I think you're good to go.
Edward Benz: I can hear you now.
Haydar Frangoul: Okay, perfect.
Edward Benz: I just got permission.
Yeah, I just got permission to unmute
and I'm going to share my screen now
[laughs].
My screen visible, everyone?
Are you able to hear me?
Haydar Frangoul: Yes, we can hear you,
and we can see your slides. You are good to go.
Edward Benz: Okay. Okay, good.
I was hearing a little bit of feedback, like echo.
But I think we can go ahead.
So thank you very much for the opportunity
to spend some time with you today.
I'm very sorry I can't be with you in person.
But I thought I'd like to just speak with you
for a few minutes about the Cure Sickle Cell Initiative
and how we're thinking about the new developments
in genetic therapies for sickle cell disease,
the fact that we now have two gene-based therapies
that are in clinical practice, FDA approved.
We're watching how those end up being utilized
in clinical practice and where we --
how we think about some of the opportunities and challenges
these developments present.
And so I'm going to very quickly summarize
an overview of where things stand now,
recognizing that you've probably heard some of this today
and you'll hear more about these therapies tomorrow,
how we're assessing the positive and negative aspects
of these new developments,
and then there -- how this NIH initiative,
called the Cure Sickle Cell Initiative,
is trying to address some of these things the future,
with the ultimate goal being not just having effective
and safe therapies available --
that's really the critical first step --
but also making sure that these therapies are accessible,
affordable by the entire community
that could benefit from them.
So as you've heard already and you'll, as I say,
hear again tomorrow in more detail,
there are two therapies available.
One uses gene editing technology in a very elegant way
to alter a control element
that in the normal course of events shut down --
shuts down fetal hemoglobin production after birth.
And by altering this control element
so that it can no longer function, fetal hemoglobin
is allowed to proceed in adult stem cells.
This therapy, developed by Crispr and Vertex,
and based on the technology and science
developed by Stu Orkin and [unintelligible]
and other colleagues, as proven in early trial results,
to be effective, so far, to have safe outcomes,
and based on a few years of experience,
to be durable so far.
The second therapy, developed by a company called Bluebird Bio,
takes advantage of the ability to synthesize the hemoglobin
that has a biochemical blocking effect
on the sickling of hemoglobin,
and thereby the sickling of cells.
And similarly to the other therapy,
it has proven to be effective so far
and to be durable so far and to have a good, safe profile.
So we have these therapies now, and that's a good development,
but as you've also heard,
the delivery of these genetic therapeutic agents,
it still requires the equivalent
of a autologous bone marrow transplant,
which carries with it many of the same risk
and disadvantages, the cumbersomeness,
and the expense of allogeneic bone marrow transplantation,
which can be curative in sickle cell disease,
as you well know, with one exception is,
of course, since this is an autologous transplant,
you don't have the issue of graft-versus-host disease.
So when you look at where we stand with this,
we have [unintelligible] that are -- look very promising.
Are they curative therapies?
Well, in our minds in the Cure Sickle Cell
Initiative -- and I think in the minds of many people,
certainly in the minds of patients
and their support systems, their loved ones, caregivers --
curative therapy is truly only curative
if it can be accessed by everyone who requires
or would benefit greatly from a cure.
And in that sense, we have a ways to go,
because this is an ex vivo method.
Right now, although you've heard some promising early research,
the only way that we have clinically
to target hematopoietic stem cells
that have to be modified for these therapies to work
is by removing them from the patient's bone marrow,
mobilizing them into the peripheral blood
and extracting them,
and then modifying them outside the body and reinfusing them
after we ablate the patient's own bone marrow
with what is currently genotoxic therapy with busulfan.
And the research you've already heard about,
and I think will hear more about tomorrow, I think,
is a critically important step in improving the accessibility
of a clinical and safety perspective of these therapies.
But there's still a lot of work to be done
to get these things into clinical practice.
There's also the complexity of the therapy itself.
There's only a few centers.
I think Vertex has authorized a number of centers
around the country and overseas,
and Bluebird, just a few that are actually able
to support the complexity of this therapy.
And it -- basically, centers
that have a high-functioning bone marrow transplant facility
and also a high-functioning cell manipulation facility,
since these cells have to be handled very much
like you would handle a therapeutic drug,
with all of the safeguards
and the additional issue of protection from infection.
Requires a long-term hospitalization.
The cost is two to three million dollars, and the way --
that is basically just for the therapy itself
and does not always include --
in the way these pricing negotiations go,
does not always include the additional ancillary
costs of hospital support and pre-hospital
and post-hospital support for the patients going through
the actual transplantation process.
Insurance coverage across the country for --
at the present time has been what I would call spotty.
Some private insurers have agreed to cover close
to all of the costs of this.
Others cost -- cover the cost of the actual therapeutic agent
but rely on whatever other systems for coverage
they have in place for the other parts of the treatment.
Governmental coverage, since most of our patients
are not old enough for Medicare or necessarily qualify
under Disability Act policies, is through Medicaid.
And Medicaid goes state by state,
and the states have been very variable so far
in how they're approaching coverage for this.
So expense is a major obstacle to generalized accessibility.
That's one of the things that we're going to take --
try to take advantage of with the Cure Sickle Cell
Initiative to meet.
We've talked about durability.
We've had a few years of observation of these therapies,
and the longest-treated patients, up to half a decade.
But we don't know, for therapy that's intended and designed --
and actually, the price is being justified
by the fact that these are one-time lifetime therapies --
we do not yet know if these therapies will indeed last
for a lifetime, if those modified cells
will function in the way we have modified them to do
for the rest of the patients' lives.
And then critically,
these therapies can stop the disease process.
In other words, the advantage of these therapies
is that they address the root cause of sickle cell disease,
which is the accumulation of this altered hemoglobin S.
But will they arrest or correct preexisting end organ damage
or the psychosocial damage or the pain
diathesis that has developed over time in these patients?
These are going to require long-term follow-up observation
and care, and our view is that just as cancer survivors
now have access cancer survivorship network
of resources at these highly sophisticated centers,
that sickle cell patients should be able
to access those very same resources where for --
the focus is on what are the clinical needs
of a survivor of a critical illness.
So if these therapies do meet the curative intent
and become curative therapies,
what about the long-term leftover consequences,
and what system will we have in place
to provide that kind of care?
Then I think there are open questions, who should get it?
In other words, when is sickle cell disease
sufficiently severe to justify the risks
and the burdens we just discussed, and at what age?
There's arguments now that the younger at which
we can treat these patients safely,
the better, before all these end organ damage
or psychosocial changes or mental health changes
and neuropsychiatric changes develop.
All that said, and all these things that represent
the less than completely perfect therapies
that these are, these are a major step forward.
They're good news for our patients,
and what we need are better ways to deliver therapeutic payloads
simply, durably, and efficiently.
And that's where the NIH
decided to develop the Cure Sickle Cell Initiative,
and I serve as executive director
and I'm presenting today
to represent the work of many colleagues
and many patients and community participants
in what we're trying to achieve.
So our vision is to accelerate the development
of genetic therapies at curing sickle cell disease
and using a patient-focused research environment
where we engage patient community
and the caregiving community in our deliberations,
in our interactions with different stakeholders
who are all trying to advance these therapies,
and really, now, with a much stronger purpose,
not so much on the development of therapies,
although we still will keep an eye on that
and support things where we can,
but really on increasing this issue of accessibility,
safety, removing financial, administrative barriers,
and so forth.
And to do that, we're going to take advantage
of some of the structural elements of this initiative
to engage partners who are usually not so easy
to involve using the NIH's usual rubrics
and rules for where you can deploy funds,
where you can seek applications for grant support, and so forth.
The whole idea is just focus on developing
these strategies for better care.
So we've focused up until now on gene
editing and gene transfer methodologies.
We are opening our window to look at small molecules
as the beginning efforts that at least some drugs
that have been reported on
in the recent American Society of Hematology meetings,
for example, that might be small molecules
that address things like fetal hemoglobin switching
and could achieve something very similar
to what the gene therapies could.
So we will look at small molecule strategies as well.
The idea of the initiative is to bring together
all these different stakeholders, as I mentioned,
with a particular emphasis on involving patient families,
people who take care of sickle cell
rather as caregivers or as medical professional providers.
And the thing this makes it different
from most of what NIH does
is this thing we call the other transactional authority.
And what -- there are a few federal agencies --
and the NIH is one of them --
out of the many, many hundreds of federal agencies
that have the ability to use this OTA methodology.
And without getting into the complexities of it,
it's a way that the government permits an agency to fund things
not necessarily strictly falling within their charter,
their sort of legislated window of things.
For the NIH, that's usually hypothesis-driven grants,
certain scientific development contracts, clinical trials,
forming databases, and some other things like that,
but not to do things that involve funding
that's shared with a private company,
for example, with other government agencies.
There's some things that are possible within NIH,
but they're quite restricted.
They're -- and they're a bit cumbersome to do.
So the other transactional authority
gives us much more flexibility
to fund things that are scientifically much higher risk
and may require partnerships that are not possible
within our usual grants and contracts mechanisms
to share information more flexibly.
Those of you who have worked within the government know
that it's sometimes remarkably hard to share
information, communicate,
and interact in real-time basis flexibly with other --
even with other government agencies
or professional societies and foundations and so forth.
And it allows us to look at multiple shots on both.
Originality is not necessarily a limiting factor here.
If somebody has developed a better way to do something,
even if it's utilizing already established --
or scientific understanding.
And it allows us also to change direction
instead of waiting until a grant expires,
that we can do things differently right away.
So to do that, we've tried to build a comprehensive approach
that would take advantage of these advantages
of the other transaction authority to fill gaps
that are not covered by other traditional funding strategies
and to fund research both within academia and the private sector
to identify the most promising therapies.
And I should point out that this is not in any way a replacement
or a cannibalization of the current NIH-funded activities.
This is mostly done through the Heart, Lung,
and Blood Institute, the initiative,
that all of the things that are in place
and all of the opportunities to receive support
through traditional NIH mechanisms are still there.
We are not touching that.
We're not taking any funds away from that.
These are supplemental funds to be creating more flexible ways
to advance toward the goal of curative therapy,
and as I mentioned, would allow us to partner
in different ways to engage the community
in different real-time ways that are done --
at least not done easily within the NIH constraints.
So here's a few of our initiative activities.
We're -- we have created a patient engagement process
that is working well through our community in [phonetic sp]
panel, and membership in all of our working committees
and in our executive committee itself,
the patient and provider representatives.
We are working on consolidating
and making more user-friendly databases
and data management, natural history studies,
and comparative controls and any other things you need
to move things through the clinical trials process,
biomanufacturing and standardization
to make the development and manipulation
these cells more efficient.
We're supporting clinical trials,
supporting outcomes research.
We're supporting a cost effectiveness [unintelligible]
through the University of Washington,
so just to give you a flavor of some of the activities
that we're going through.
And this is just gives you a quick peek
at some of the partners that we've developed over time.
You see, these are things not possible
within the usual NIH rubrics and rules --
working with professional societies,
working with the Stem Cell Institute in California,
working with CMS on coverage and payment issues, et cetera.
So I want to finish by just telling you what we're hearing,
and I don't claim that we are hearing everything
or that I can cover everything that we're hearing in this talk.
But from engaging with the patient community
and through focus groups,
through the work in real time on our subcommittee, these --
through the development of materials
and our online interactions with the community,
and also with traveling around [inaudible]
of the country -- this map is out of date.
We've gone to the west coast now,
not quite as confined to the east coast.
This started during the pandemic when overnight travel
was a bit more of a challenge,
but we've been pretty much around the country now,
engaging groups and hearing what patients want to say.
And this is what we're hearing.
Most families and patients are quite excited
to have these treatments available,
want to get them if they can,
want to know if they can be able to afford it or access it.
We've talked about some of the challenges there.
Very strong emphasis on if I go through all this, will it last?
Will I be trading the dangers of sickle cell disease
with the -- for the dangers of leukemia?
Now we know that the chances
of developing myelodysplastic syndrome or leukemia
are quite small in the trials that we've seen so far,
but they are known to -- by the patient community,
and of course, because of the nature of the disease
that -- being discussed, that is a concern for patients.
So -- and we've heard really great communication
with patients about the pros and cons,
the risks and benefits of going forward
with these therapies is important.
And I'll finish up by telling you some other efforts
we're trying to make to see
if we could make the prediction of those risks more accurate.
Will a cure affect my identity?
I'm always reminded when I've seen a patient
with any disease who has suddenly --
been living with that disease for a long time
and suddenly is no longer dealing with it
that there is a bit of an identity crisis.
This is well known in the community of deaf persons
who have their hearing restored,
because they have lived in a community.
They've lived within a support system
that find the boundary conditions of their life
are around their chronic illness,
and if that is suddenly altered,
it requires a really significant reset of one's sense of self
and one's aspirations and ambitions
and capabilities to achieve those might be.
So, what's life without the disease going to be like?
We've heard a lot of that in talking to our patients.
And then of course, the blood picture,
they can expect to be better,
but what about all the other chronic problems,
the end organ damage, the pain,
any of the preexisting neuropsychiatric damage
that this disease has done to them?
And those are still open questions.
And then will I get the follow up I need?
Will I lose my care team?
Those patients fortunate enough to be getting their care
at good sickle cell center or sickle cell clinic
worry that they won't be able to get that.
Now that turns out not to be true,
that people are not going to abandon these patients,
but it is a legitimate question for people to raise.
And that's where I come back the notion of a survivorship program
that we're trying to help develop
for our patients who undergo these therapies.
So here's what we're -- finally, what we're funding.
We're open to input from people who are trying to develop
simpler, safer, cheaper delivery.
We've heard some presentations in that today,
maybe some more tomorrow. Small molecule drugs,
which clearly would be simpler to deliver.
So we are having a workshop coming up in October.
We're going to be hearing from people working in this field
and seeing if there's ways that we can use our funding mechanism
to accelerate the advances in this early-stage research.
Also, similarly, for non- genotoxic conditioning regimens,
because in the meantime,
while all these other therapies may be developing,
patients will be getting bone marrow transplants.
They will be getting ex vivo gene therapy.
And we -- as soon as possible,
we'd like to see if we can accelerate the development
of some of the genotoxic conditioning regimens
that you just heard about a couple of presentations ago.
We are developing this long-term follow up.
This includes survivorship studies, mostly to collect data,
but also to develop programs
that we might incent people to develop at these care sites
to manage long-term survivorship issues.
We have developed national cell bank resource,
so you can get bio samples for doing some of these studies
on targeting hematopoietic stem cells, for example.
And we're funding a study
on so-called clonal hematopoiesis
of indeterminate potential.
This is a factor that we all have in our bone marrows,
and as we age, we develop mutations
in our bone marrow stem cells
that increase the risk of a number of illnesses,
including the likelihood of hemalogic
[phonetic sp]
malignancy, to see if there are ways to identify
those individuals whose so-called CHIP situation
may be more protective or more high risk
in undergoing these therapies.
And then we are using cost effectiveness analysis,
our ongoing study of that, to make the case with CMS
and with private insurers that they should be covering
this care more comprehensively and more completely
and with a better reimbursement system
that doesn't put the provider institutions
at risk of having to collect the reimbursement
after paying $3 million per patient to start the therapy.
So that's where we stand, and we're here today
mostly to make you aware of what we're trying to do
and welcoming your input.
Just use our website, the Cure Sickle Cell
Initiative -- or the curesickle.org,
and we will keep you apprised of where what you're working on
and what we're working on my align
and how we may engage together in making this situation
even more promising for our patients and their families.
So thank you very much.
I'll be happy to take any questions.
[applause]
Haydar Frangoul: Thank you, Dr. Benz.
I will entertain few questions from the audience,
but I want to -- can I add something to your list
that you put in there myself on your cure sickle cell?
Can I add a big plug in for fertility preservation
for individuals with sickle cell disease
going through gene therapies and --
Edward Benz: Yes.
Haydar Frangoul: -- curative therapies?
Edward Benz: I'm so happy -- thank you. Thank you.
I'm so happy you said that. I meant to tell you
that we are working very hard on that from two perspectives.
You know, one is clearly better technology
that would make fertility loss less of a risk,
but the most real-time thing is we're working with CMS
to make sure patients are covered for those treatments.
And that's another reason to try to take advantage
of the survivorship system
that's in place for cancer patients.
Cancer patients in many states, not all,
but a fairly large number of states
can get coverage for fertility preservation
for their chemotherapy,
but we're not able to get it for our patients
undergoing bone marrow transplant
or therapies for sickle cell.
In some states you can, but very few.
So we are working with CMS and private insurers on that.
Haydar Frangoul: Yeah.
I personally don't think a patient should be choosing
between a cure and having a family.
They should have both. But that's my point.
Edward Benz: I completely agree.
Haydar Frangoul: Any questions from -- okay.
Jane Little: This is Jane Little from UNC.
Thanks, Dr. Benz. I had a question.
In hemophilia and cystic fibrosis,
the long-term follow up is embedded
within their own longitudinal registries.
And I -- and that seems like a good model for sickle cell.
I wonder what your thoughts are about that.
Edward Benz: Yes. I agree, and that's one aspect
of long-term follow up that I think,
although we're working with the usual NIH HLBI efforts,
is that they are doing that.
They put together under the rubric
of a natural history study that they're --
what they're trying to do is coordinate the databases
so that the long-term follow up data can be there.
We're trying to work now on a program
to make sure that people who are not necessarily participating
or contributing to those databases,
if these patients undergo these therapies,
that they will be supported to put the necessary data,
you know, to use the right templates,
to enter the right data fields in and standardize
those as best we can.
The survivorship model I talked about focused
more on the actual delivery of the care
as well as gathering the data.
Jane Little: Think the consistent templates is --
that's a really important feature. Thanks.
Edward Benz: Yes, I agree.
Haydar Frangoul: Well, I would like to thank all the speakers.
If there are no questions,
I think there are some closing remarks going to be given.
Monika will be giving the closing. Thank you.
Monika Asnani: Closing remarks are really short [laughs].
We have had a long, wonderful days.
Long, so we are tired, but I must say,
I'm sure you all agree, we've had a fabulous day.
And it's going to be even better tomorrow, if at all possible.
[applause]
So thank you to all the speakers for working so hard, Dr. Benz,
and all the chairs. Thank you to the admin team.
[unintelligible] because really --
[applause]
-- working across two different regions.
We'll do a bigger thanks tomorrow, but thanks, all.
Go and have a good evening. Enjoy. Rest.
Go to the pool, those of you who want to.
Go get some food, and get some rest.
See you all tomorrow bright and early.
Sorry? Oh.
The speakers are to meet -- where, Nancy? At the lobby?
Female Speaker: Yeah.
Moninka Asnani: By 6:30 p.m.
Those of you who have a speakers' dinner --
6:15 p.m., sorry. You meet downstairs at 6:15 p.m.
so you can reach your venue on time.
And see you all tomorrow. We begin sharp at 8:30 a.m.