A study published in The New England Journal of
Medicine in December 2011 highlighted a potential breakthrough in gene
therapy, documenting how British researchers used the technique to successfully
treat six patients with hemophilia B.
Katherine A. High, MD, the William H.
Bennett Professor of Pediatrics at the University of Pennsylvania School of
Medicine, as well as an attending hematologist and director of the Center for
Cellular and Molecular Therapeutics at The Children’s Hospital of
Philadelphia, led a round table discussion during the HemOnc Today
section editors’ retreat April 27-28 to further explore the study results,
their potential implications and the future of gene therapy in hemophilia B.
A transcript of that round table discussion follows.
Dr. High: Hemophilia B has long been a goal for
people interested in gene transfer. The reason for that is, most people with
the disease have circulating levels of Factor IX of less than 1%, but if you
could just raise the levels to the range of 5%, you would convert the
person’s disease from severe to mild. This has been easy to do in mice and
hemophilic dogs but surprisingly difficult to do in humans.
Katherine A. High, MD, director of the Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia, said it remains unclear whether emerging therapies will have ramifications for the 80% of the world’s hemophilia population who currently do not have access to optimal treatment.
Photo courtesy of Katherine A. High, MD, reprinted with permission.
The first trial, in which this kind of vector was
infused into the liver, was reported in 2006. The patients at the highest dose
got therapeutic levels of Factor IX in the range of 10%, but they only lasted
for about 6 weeks and they disappeared at the same time that the patients
developed an elevation in liver enzymes.
Results of a more recent trial, jointly sponsored by St.
Jude Children’s Research Hospital in Memphis and University College London
and the Royal Free Hospital in London, were published in December in The New
England Journal of Medicine.
One of the important changes to the protocol in this
trial was that they stipulated that if they saw a rise in liver enzymes or a
drop in the Factor IX level, they would start the patient on high-dose steroids
and give a course of steroids for 4 to 8 weeks to try to protect the patient
from what appeared to be an immune response to the viral vector capsid and
rescue the transduced cells and keep them from being destroyed by the
patient’s own immune system.
Dr. High: Just to backtrack, what they observed
in the first trial was something that looked a little bit like an autoimmune
hepatitis but, in fact, it was alloimmune hepatitis. It was a patient mounting
an immune response to the vector capsid.
In the trial in London, in the first two dose cohorts,
there was long-term expression at low levels around 2% of normal, but for
people with hemophilia, moving from less than 1% to 2% is clinically meaningful
in terms of how much they need to rely on clotting factor concentrates. But in
the high-dose group, the patients initially had levels of around 8%, and that
would really make them mild hemophiliacs, and that would be a very important
One of those people did develop this transaminase
elevation. It was unclear at first what was going on because it happened much
later than had been observed in the previous trial, so there was some confusion
about whether this was something that should trigger the steroids, but he was
eventually put on that. He leveled out at around 2%.
The other patient in the high-dose cohort ended up with
a level of about 6%, so he is a very happy individual now. He apparently plays
rugby on the weekends, which is an unusual leisure activity for someone with
severe hemophilia, but I guess he used to dose himself with factor before he
played. Now he doesn’t have to.
This is a very important landmark for people with
One of the ironies of this is that it is coming to
fruition at the same time that the major manufacturers of clotting factor
concentrates are in the late stages of investigation with new, longer-acting
clotting factor concentrates. So it is expected that these will go to licensing
within the next year or two.
The half-life of these new, longer-acting Factor IX
products is on the order of 90 hours, instead of the 12 to 24 hours of
currently available clotting factors, and it is expected that patients will
only need to dose themselves about twice a month.
The question is: Where will these different types of
therapies fit in, and will they have any ramifications for the 80% of the
world’s hemophilia population who currently do not have access to optimal
treatment? The treatment we use today, infusion of recombinant protein, costs
about $300,000 a year if you do prophylactic infusion in an adult male.
Consequently, people in North America and Western Europe are managed that way
but, in most other countries, the consumption of factor is a good deal less.
How will these new therapies go forward? Will these
results be replicated, and can they be extended, for example, to Factor VIII?
Those are some of the questions that people in the field are thinking about
Costs are unclear
Dr. Green: Is there an estimate about whether the
new, longer-lived forms will be more expensive?
Dr. High: I would expect, based on previous
experience, that the manufacturers will advance the opinion that they’ll
be making a better product available for a similar cost so the total yearly
cost to the patient would not rise, but it would be easier and more convenient
for the patient.
Dr. Green: What is the cost of the adenovirus
Dr. High: Because there are no adeno-associated
virus (AAV) products on the market, it is very difficult to predict. The cost
of goods for the manufacturer of recombinant AAV goes down every year. At
Children’s Hospital, we have a trial running for an eye product and, of
course, the doses of that are logs lower than for a hemophilia product, but I
know in that case the cost of goods is less than the cost of a single dose of
clotting factor in an adult male. I believe the cost of goods will not be the
prohibitive expense, but, of course, there’s a lot that needs to be done
in terms of development of these promising results.
Dr. Bertino: Have they really looked at the
integration sites and it is also in bone marrow stem cells or just liver cells?
Joseph R. Bertino
Dr. High: This vector is infused intravenously,
so the infusion procedure is easy and convenient for the patient. AAV is
stabilized predominantly in a non-integrating form, so in terms of what the FDA
and other regulatory agencies expect about analysis of integration sites,
it’s less challenging than it is for retroviral and lentiviral vectors,
which rely on integration in order to work. However, as you have mentioned,
whenever you put foreign DNA into a cell, you run some risk that there will be
integration events, and at least in animal livers, this has been observed with
the types of doses that are being used in the hemophilia trials.
For the subjects who have been enrolled on these trials,
liver biopsy is not part of the trial protocol, so there has been no analysis
of the target organ in human subjects. In terms of vector shedding in the body
fluids, that’s been followed and all of the body fluids have been positive
for some short period of time after the vector infusion, but nothing is
Dr. Boxer: One-third of the adult population has
immunity to adeno-associated virus, so what’s going to be the outcome of
these patients, and are other vectors being developed?
Dr. High: Most of us have been exposed to the
wild-type virus from which this vector is engineered, usually during childhood.
It doesn’t cause any known illness, but something like 30% of the
population has circulating antibodies to AAV, and if you try to infuse the
vector through the bloodstream, those antibodies are powerfully neutralizing
and those people won’t get a therapeutic effect. Right now, those people
are excluded from trials that rely on intravenous infusion. Studies have looked
at plasmapheresis as a way to get rid of the antibody transiently, and there
are drugs, such as Rituxan (rituximab; Genentech and Idec Pharmaceuticals),
that can get rid of B cells. Those issues will need to be addressed in further
studies, but right now those people are excluded from the trials. That’s a
big proportion of the population, so people will be motivated to figure out a
way to include them.
Dr. Jacob: In the presentation of your group at
the American Society of Hematology Annual Meeting in December, I got the
impression you are using an adeno-associated virus 8 now. My impression was
that very few people actually have been exposed to that, whereas your previous
studies were with adeno-associated virus 2, which was a common problem of
immunity. Have I got that wrong?
Harry S. Jacob
Dr. High: Between the first trial and the second
trial, a different AAV vector was used, a switch as you said from serotype 2 to
serotype 8. The prevalence of neutralizing antibodies in the population is
lower for 8, but it is still fairly substantial. These capsid structures are
pretty highly conserved from one serotype to another, so the antibodies
cross-react fairly extensively. Even though it’s a lower number than for
AAV 2, it’s still fairly substantial. The other realization has been that,
we thought at first that only fairly high titers would prevent transduction and
it’s become clear that even modest titers will prevent transduction.
That’s part of the reason for that change in numbers of how many people
are prevented from getting the therapy by the antibodies.
Impact of hepatitis
Dr. Rao: This presentation at ASH was a seminal
event from the perspective of patients who have hemophilia and for the entire
field of gene therapy. It made the point that we can achieve gene therapy, and
it can be effective.
One specific question: Many of these patients are
positive for hepatitis C virus (HCV) or hepatitis B virus. The older patients
probably have a higher incidence, and this has been a limitation in current
trials in selecting individuals for gene therapy. Is that going to be an issue
and, if it is, how would that get handled?
Dr. High: That’s a very good question, and
it’s a difficult issue for the trials. In the first trial, using AAV2,
because we had not expected this alloimmune hepatitis, we enrolled subjects who
were HCV-positive so they could be HCV RNA viral load-positive and still enter
the trial, and no adverse events were seen, including in these people. Now,
because we know that a certain percentage of people who get that high dose are
likely to need a course of prednisone for 4 to 8 weeks, HCV RNA viral
load-positive patients are excluded because people are worried about putting
them on steroids for 4 to 8 weeks. The question is whether it will be possible
to fashion a regimen that will be effective against this transient immune
response against the capsid but that will be safe for people who are HCV RNA
viral load-positive. What we think, after conferring with hepatologists, is
that for individuals who clear on the new anti-HCV regimens, it will be
possible to allow these people to enter the trials.
Dr. Rao: There will eventually be a place for
both the newer products that are coming down the pike, as well as gene therapy,
simply because there will be a huge population of hemophiliacs who will not be
candidates for gene therapy. There will be an indication for both.
A. Koneti Rao
Dr. High: Yes, and your question points to
something important. There is success in a well-defined patient group, but it
enables us to both extend that result and begin to work on some of these other
problems. What do you do about people who have antibodies to AAV? What do you
do about people who are HCV RNA viral load-positive and can’t go on
steroids? Is there another regimen that you can give them? A lot of these
people go to transplant. They get immunosuppression. There should be a regimen
that is usable in that situation. We just have to figure those things out.
Factor IX vs. Factor VIII
Dr. Coleman: Why did you choose Factor IX instead
of Factor VIII?
Dr. High: The AAV vector can accommodate a
transgene of about 5 kilobases (kb). For Factor IX, the coding region alone is
about 1.5 kb and the coding region of even B-domain deleted Factor VIII is 4.4
kb, so you can put it in there, but you have to engineer a very small promoter
and a very small intron and so forth to get that Factor VIII in.
Dr. Rao: Most hemophiliacs have Factor VIII
deficiency, not Factor IX deficiency. Because there are limitations based on
the size of Factor VIII molecule, there are some alternative methods that have
been proposed. One is using megakaryocytes and platelets to deliver Factor
VIII, driven by a platelet-specific promoter, in which case they are actually
delivering Factor VIII to the site of the bleed, since platelets accumulate
there and release their contents. Moreover, should gene therapy use Factor
VII-a expression to correct the hemostatic abnormality given that expressing
Factor VIII has had some limitations?
Dr. High: It has been possible to make an AAV
vector that expresses Factor VIII, and I suspect you will see a clinical trial
of that begin within the next 2 years. In terms of the other strategies that
you discussed, I’m a hematologist, so I was always attracted to the idea
of trying to put one of these genes into a hematopoietic cell, including a
megakaryocyte. What prevents people from moving to that right away is that, for
all of the strategies so far that rely on genetic modification of autologous
hematopoietic cells, you have had to do some sort of myeloablative conditioning
to make a niche for the transduced cells. That has been unattractive to
hemophilia patients. Everybody is still looking for a solution, medicine in a
bottle that you can take down off a shelf and inject and you’re finished,
and bone marrow transplantation is a little more involved than that. However, a
good niche for that strategy may be, as you mentioned, inhibitor patients who
have failed on immune tolerance induction because, as you say, if you can
package the Factor VIII into granules within a platelet and they release the
granule contents at the site of a bleed, you may have a way to get around an
inhibitor. I wonder if that’s the place to begin with that strategy.
Dr. Rao: How about the approach expressing Factor
Dr. High: We have done a lot of work with VII-a.
The challenge for moving that into the clinical arena will be that it is likely
that you are going to need a regulatable promoter, so it would be a promoter
that can raise the levels of VII-a up or down based on an oral drug. The reason
for that is, although it’s all right to have constitutive expression of
Factor VIII or Factor IX, people may not feel the same way about constitutive
expression of Factor VII-a.
Future of gene therapy
Dr. Gordon Smith: Where are we going to be 2
years down the line in terms of gene therapy?
Dr. High: It’s difficult to answer that
question. There are no licensed gene therapy products at this point in ICH
countries, so the regulatory pathway is not entirely clear. There are products
that have been through phase 3 or that are approaching phase 3. It may be that,
within a couple of years, we will at least have a road map of what is required
for licensing. The product we use to treat Factor IX deficiency was licensed on
a trial of around 50 patients. It’s possible that large trials won’t
be required, but until there are licensed gene therapy products, it will be
difficult to comment on that.
Dr. Jacob: You talk about the immune suppression
of your vector, which you evidently can handle fairly readily. Are there other
suspicions that, without an immunologic attack, your gene is just going to
peter out anyway? Is there any evidence that occurs in animal trials?
Dr. High: The data in dogs have yielded a
surprising finding. Ten years after an injection, these hemophilic dogs are
still expressing Factor IX at the levels that you observe at about 6 weeks when
they hit a plateau. I find that very surprising. I would have expected the
levels to slowly decay. It will be important to see what happens in these first
subjects, whether they maintain the levels for 2 years — which they seem
to have done already — or longer than that, and whether re-administration
is going to become an issue. For that reason, what people would like to do is
be able to give enough vector to get an initial level in the range of 20% to
40%, so you’d have a comfortable margin even if there were a slow decline.
Dr. Coleman: One of the reasons you’re so
successful is the liver cells just don’t turn over as rapidly as
hematopoietic cells because it’s been tried. They tried to put in the
NDR1 gene so they could deliver high doses of chemotherapy, but they
were never very successful. The plating efficiency was poor. Liver cells, if
you have damage, can divide, but I gather they don’t turn over so quickly.
Do you know the half life or mitotic rate?
Dr. High: I have tried to find out from
hepatologists what they expect the life span of a hepatocyte to be and I have
not been able to find that out. You are right, it could be addressed
Dr. Coleman: It would be very interesting to see
if the daughter cells also carry the gene.
Dr. High: Certainly based on animal studies, we
believe the daughter cell will not carry the donated gene, but then I cannot
explain why dogs would still have the same level 10 years later.
Dr. Coleman: You might want to consider taking
out a large portion of the liver from the dog and let him regenerate and see
what happens. I don’t know whether you’ve done those experiments.
Dr. High: We’ve done those experiments in
mice. What happens then is, if you’re sitting at about 100%, you remove
80% of the mouse’s liver. As the liver regenerates, you do not get the
gene passed to the daughter cells and the levels go very low.
A more affordable alternative
Dr. Jacob: Assuming that one ultimately could get
this therapy approved, if this is one intravenous infusion of a product, would
this not be an incredible situation for poor countries — one injection
compared with no possibility of the $300,000-a-year kind of recombinant type of
Dr. High: If the data continue the way they are
going now, this could be a situation where you could make a single intervention
and then return the patient to his home setting. He would not need a great deal
of follow-up and he would be much safer than he is now if he is a person who
bleeds and has to solve difficult transportation issues to get to a place where
he can get the factor. It seems to me that it would certainly have a great deal
to offer for that setting. The questions are: What will be the availability,
who will control the distribution and what will the cost be?
Dr. Rao: My view is that, in most developing
countries, it will be difficult to afford the annual costs of the replacement
with recombinant products. If gene therapy is established, and I think it will
be, that will be the way for most of those countries to go rather than depend
on recurrent use of products.
Dr. High: I don’t disagree with you. This
has been a complex product to manufacture. It has a protein coat with a DNA
sequence that is the active agent, so entities that want to develop the product
are going to have to become expert at a fairly complex manufacturing process,
but obviously there are individuals everywhere who can do that.
Katherine A. High, MD, the William H. Bennett
Professor of Pediatrics at the University of Pennsylvania School of Medicine,
as well as an attending hematologist and director of the Center for Cellular
and Molecular Therapeutics at The Children’s Hospital of Philadelphia, led
the round table discussion.
Other participants included Ralph Green, MD, a
professor in the department of medical pathology and laboratory medicine at UC
Davis Medical Center; Joseph R. Bertino, MD, associate medical editor of
HemOnc Today; Laurence Boxer, MD, professor and director of
pediatric hematology/oncology at the University of Michigan Medical Center;
Harry S. Jacob, MD, FRCPath(Hon), chief medical editor of HemOnc
Today; A. Koneti Rao, MD, Sol Sherry Professor of Medicine and
section chief of hematology at Temple University School of Medicine; Morton
Coleman, MD, director of the Center for Lymphoma and Myeloma at Weill
Cornell Medical College; and Edward Gordon-Smith, MD, MSc, FRCPath, a
professor at St. George’s Hospital Medical School in London.
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