Round table conference in Monaco on 19 June 2004:
The second Monaco round-table
conference in 2004 was organized by the Association Monégasque Contre les
Myopathies and the Duchenne Parent Project France, both of which belong to the
international United Parent Projects Muscular Dystrophy, UPPMD.
Twenty scientists from six
countries met in Monte Carlo on 19 June 2004 and discussed their work on the
transfer of the full-sized and shortened versions of the dystrophin gene into
the muscles of Duchenne boys.
The conference was opened by Prince
Albert of Monaco who welcomed the participants and said that “Monaco will
always be an open meeting place to all who will give children hope for a better
life, especially for those with such a disabling disease”.
This report will concentrate
on the first clinical trial of a gene transfer technique with Duchenne patients
now underway in France and on others planned for the near future. This
information is based mainly on two interviews which were conducted immediately
after the conference. They are preceded by an introduction to the transfer
techniques involved. A conclusion based on comments from Professor George
Karpati then brings the report to a close.
As the interviews mainly tried
to answer the question always asked by the parents "How long do we have to
wait, until a therapy will be ready for our boys?”, this report was written
mainly for the families and their pediatricians and not for scientists who wish
to be informed on the details of gene transfer research for muscular dystrophy.
In addition to the
presentations and discussions about the clinical trials with the gene transfer
technique, there were a number of others which, because of the missing records,
cannot be included here. They dealt mainly with the details of the gene
transfer techniques like the advantages and disadvantages of different vector
constructions and the problems of immune reactions against the vector and the
newly synthesized dystrophin.
A comprehensive research
report on the state of Duchenne research as of August 2003 can be seen on the
internet at http://www.duchenne-research.com. This report will be updated
probably in 2005 and will then include information on all approaches toward a
cure of Duchenne muscular dystrophy including the work which could not be
mentioned here.
Duchenne muscular dystrophy is
caused by mutations of the dystrophin gene on the X chromosome. This gene is
the largest gene found in the human genome. It is about 2.5 million base pairs
(genetic “letters”) long which are grouped in 79 exons with a total of only
11.000 base pairs, its active regions, that contain the information for the
production of dystrophin. This protein stabilizes the muscle cell membranes. If
a mutation of the gene changes the genetic information in such a way that no
dystrophin can be made in the muscle cells, Duchenne muscular dystrophy
develops. However, some mutations do not interrupt the dystrophin production
but only cause the formation of shorter than normal dystrophin. This leads to
the milder symptoms of Becker muscular dystrophy.
At
the first round table conference, exon skipping was discussed as a possible
technique to change a "Duchenne mutation" into a "Becker
mutation" by instructing the protein synthesis mechanism not to use
certain exons with the aim to produce a shorter than normal dystrophin instead
of a complete stop of its production. With the techniques discussed at this
second conference, one tries to transport the combined exons, the cDNA, of
normal dystrophin into all muscle cells with viruses or plasmids as carriers,
called vectors.
The most effective viruses for
this task are the adenoassociated viruses, AAV. But these small viruses can
only transport genetic material that is not longer than about 5,000 base pairs,
about one third of all the exons with the information of dystrophin..
Their advantage is that they
transfer the gene more effectively than other viruses like the normal
adenoviruses. The disadvantage is that the dystrophin cDNA to be transferred
has to be shortened considerably to fit into this small vector together with a
promoter sequence for activating the gene in muscle cells. Patients with Becker
muscular dystrophy have similar shortened dystrophin in their muscles.
Therefore, a transfer of one of these Becker minigenes might not completely
cure Duchenne muscular dystrophy, but only transform it into the benign Becker
form.
The shorter forms of the
dystrophin gene are called mini or micro dystrophin genes, depending on their
structure which might be as short as one third or more of the natural length.
Although the title of this conference suggests that only micro dystrophins were
discussed, in fact the transfer of mini and even full-length dystrophin genes
were subject of some presentations.
The first interview was
conducted with Dr. Serge Braun, Vice-President Research of the biotechnology
company Transgène in Strasbourg, and Dr. Jon Wolff of the University in
Madison/Wisconsin and President of Mirus company. Its subject was the first
gene transfer trial with Duchenne patients, in which not viruses, but plasmids
were used as vectors to transport not a shortened version of the gene but the
entire combined exons, the cDNA of the full-length dystrophin.
The first phase of this trial
was completed at the beginning of 2003. The company Transgène and the
French muscular dystrophy association AFM started their research and
development program in 1995. The permission by the regulatory authorities was
given in November 1999, and the first injections of the plasmid vectors with
the dystrophin gene were performed in September 2000 at the Hôpital de la Pitié
Salpêtière in Paris.
The 9 participating boys were
all older than 15 years so that they could give their informed consent. They
did not derive any clinical benefit from this treatment, it was not yet a
therapy. Its aim was to show that the procedure is safe, i.e., that it does not
lead to an immune reaction or an inflammation, and that some new and normal
dystrophin appears underneath the membrane of the fibres in the treated muscle
tissue.
For this technique, the
combined 79 exons without the introns, the cDNA, of the normal dystrophin gene
and its controlling structures were part of the genetic material of plasmids.
Plasmids are small circular DNA structures without protein inside bacteria to
which they mostly confer resistance against antibiotics. As the plasmids do not
contain any protein, only genetic material, naked DNA, no immune reaction
develops against this vector and its charge.
In preliminary experiments
with muscle cell cultures, dystrophic mice, and dogs, it was shown that this
vector construction led to the appearance of new dystrophin at its correct
place underneath the muscle cell membrane of the animals, that it restored the
dystrophin-glycoprotein complex and that it prolonged the life of the cells.
The amount of plasmids injected into the animals was proportionally up to 500
times greater than that used for the Duchenne patients in the trial.
A solution with 0.2 mg
plasmids containing 10 trillion (10 x 1012) copies of the dystrophin gene was
injected into one muscle of the forearm of the first three patients. The next
three patients received one dose of 0.6 mg and the last three two doses of 0.6
mg two weeks apart. Each patient was treated only when it was certain that the
previous one treated did not show any signs of immune intolerance or other
problems.
Three weeks after the
injections, the treated muscle volume of about 0.5 cubic centimetres was
extracted by biopsy and checked for the presence of dystrophin. In three out of
six patients in the first two groups and in all three patients in the third
group, new dystrophin appeared in less than 1% to more than 25% of the muscle
fibres around the injection sites. There were no signs of an immune reaction,
neither against the plasmid nor against the newly produced dystrophin. This
answered the question of a phase-I study: Gene transfer with naked DNA is a
safe procedure.
The French scientists are now
working with the team of Jon Wolff in Madison/Wisconsin, who injected similar
plasmid constructions with genes of a marker protein into the blood stream of
limbs of rats, dogs, and monkeys under pressure produced by short-term blocking
of the blood circulation in one limb. Afterwards, up to 40% of the muscle
fibres contained the transferred marker protein.
When
relatively large volumes of a solution of these plasmids were injected under
pressure into the veins or arteries of the limbs of the animals, the reporter
genes beta-galactosidase and luciferase were transferred into up to 20 % of the
muscle fibres after one single injection and into up to 40 % after repeated
injections. The pressure was created by blocking the venous outflow from the
limb with a blood-pressure cuff for a short time. The French and American
researchers have joined their forces to take this procedure to Duchenne and
Becker patients. Encouraging results have been generated in the Golden
retriever muscular dystrophy dog.
The questions of this and the
second interview were asked by the author of this report, Dr. Günter
Scheuerbrandt, they are printed in italics.
In this discussion, we should try to find
an answer to the question the parents with a Duchenne boy always ask: How long
will it take until the gene transfer method will be available to cure our son?
In an interview at the last Monaco meeting in January on exon skipping, the
answer was: ten years, more or less.
B: It is quite impossible to answer this
question. Usually with any new drug, it takes about 15 years to go through
phase I, phase II, and phase III of a clinical trial. With a genetic disease,
it might be different because we could possibly obtain an orphan drug status
and a fast track approval, especially for a disease like Duchenne with no
treatment. But even then, it will be a long way. With our plasmid DNA study, we
just completed the phase-I trial, which means we went at least one-step
forward.
How long did this trial last?
B: Two years and a half plus 18 months of
discussions with the regulatory authorities, that is four years just for phase
I. But it took so much time also because it was the first gene-based trial for
Duchenne dystrophy. So, there were more issues to discuss, and more questions
and more concerns were raised by the regulatory authorities before any approval
was given for clinical trials.
Are these regulations in France different
from the ones in the United States?
B: I think, they are similar and very
strict in both countries. The French authorities are really very cautious about
genetically modified organisms, GMOs, and plasmid DNA is considered as a GMO
like modified viruses.
Now, the first phase has been finished
for Duchenne dystrophy. Were the regulatory authorities content, did they
accept the results?
B: There is still a lot of work to do
once the trial is finished. We have to gather all the data and have it prepared
by following very strict rules, and to write a report for the regulatory
authorities. So we will go to them this fall, probably in October.
Now, for the first phase, did you
actually use the pressure method which was developed in the US?
B: No, because we have to proceed step by
step, and the first step was local administration of a low dose. Nothing bad
happened. But it was really not a treatment, and brought no benefit to the
patients. For the clinical development of a new drug, that is the best way to
go. Phase I is to check for toxicity, safety, and tolerance. If the results are
o.k., then you can move on to a study in which you want to see some benefit.
That would then be a phase-II trial?
B: Yes, but in our case, it is a
phase-I/II trial, because we changed the protocol as we will switch from local
administration to local regional administration by intravascular delivery. And
this will be done with Jon Wolff’s method. I remember when I visited you, Jon,
in 1991 with a father of a Duchenne boy. During the discussion, you stepped out
of your office and came back with just a syringe and said “that is all we
need”.
W: That was the starting point. And even
now we basically need only a tourniquet, a needle, and a pump. We tried
injections into veins early, but with a big vein, we had a valve problem. With
our present technique, that goes back to 1995/96, the administration is done
into small veins, it was developed at my company Mirus by Jim Hagstrom and
Julie Hegge. In the phase-I/II trial, we will use this method.
Nine children participated in
the first trial who were 15 years old at least because they had to give
informed consent. How will this be in the next phase, can you work with younger
patients?
B: We wish to work with younger patients,
and we will ask both the American drug agency FDA and the French drug agency to
allow us to enrol younger patients. But first we have to accumulate more data
and then have pre-meetings with them to explore this possibility.
How much time will you need to prepare
the next phase?
B: It is difficult to say, because it
takes several months to get an answer from the regulatory authorities, usually
about three months. In our case with the first phase, it took 18 months.
Is there a reason why it
takes so long? There are important people in these committees. Are they not
interested? This is a disease that is just awful, it is terrible. And the
children are dying away, the family’s time is running out. Is there just red
tape, bureaucracy, that they cannot work faster?
W: They are understaffed, at least in the
FDA.
B: And in France it is not different.
But in general, these people are
interested that trials are being done?
B: They just don’t have the experience
and they are not alone, because this is a completely new field of research.
They have to learn, we have to learn. We have to do the trials in a very safe
way, and their role is to assure we are doing it very safely. That is why they
take so much time and precautions and raise so many questions. And this is for
the patients’ sake.
At the meeting in
January, Nick Catlin who represents the British Parent Project, said quite
emotionally what the parents really want: Even if there is risk, the scientists
should go ahead.
W: That is not the right way to do
things.
B: If anything happens during such a
trial, it would do a lot of harm to the whole field, it would stop everything.
So we have a huge responsibility. And that was also what we felt with the
phase-I trial: Had anything negative happened, that would have been detrimental
to the whole field of gene therapy.
W: Clinical trials are very expensive and
time consuming, so you want to do it right, you need all the information you
can think of.
You said that you have spent already 25
million dollars for the first trial. Has this being paid completely by the AFM?
And your company, Transgène, must have provided something, too.
B: This amount was necessary for the
whole trial but also for research, and for the salaries of the people working
on this project. The AFM paid this completely and continues to pay for these
trials. Our company Transgène provided know how, manpower, equipment,
expertise, and scientific development. All this is highly valuable. We have
three-year contracts with the AFM, the present contract ends at the end of June
of this year. And as usual, every year, we provide them with complete reports
and have meetings with the scientific committee. On a regular basis, they
verify all the expenses. Transgène is a public company. So these
financial matters are open to everybody.
If it costs so much money for the
development, then how much will the treatment about cost to the parents? Will
it be expensive?
B: Probably. But there is one example:
Gaucher’s disease. It is a rare disease, and the treatment with the missing
enzyme is very very expensive, 300,000 dollars per year. It is covered by
health care. We do not now how much the Duchenne treatment will cost, but it should
not be as high,
How big are your companies, and how many
people are working on this project?
B: Transgène has 165 people, and
about 30 of them are working on Duchenne gene therapy, some of them part-time.
W: And at Mirus and at the university in Madison there are about 6 people
working with me.
To come back to the next
phase of the trial: When will you be able to start and how long will it take?
B: It will take about a year for the
preparation, and when the next phase is approved, we will probably need as much
time as for the phase-I trial. That lasted two years and a half, maybe we can
do it quicker this time. But it all depends on the regulatory authorities. If
they ask again to enrol patients on a sequential basis, then it will take two
and a half years. But again, that would be for safety reasons. And there is
nothing we can do about.
And how many patients will take part in
the phase-I/II trial?
B : It is still a matter of discussion.
Maybe 15 patients.
Our families will say: Oh, we take our child
and go to Strasbourg as others went to Memphis to Peter Law.
B: All the patients will probably be
American and French boys. Nevertheless, this remains still open. But we are not
responsible for deciding whom to enrol in the trial. Only clinicians have the
right to make those kinds of decisions. The principal clinical investigator in
France will probably be again, as for the phase-I trial, Professor Michel
Fardeau. The new trial will also take place in the US, but we do not yet know
who will be the principal investigator there.
Will you start in the two countries at
the same time? Will you use the same method, will you just have more patients?
B: Probably. We will ask the FDA and the
French drug agency and we hope that the trial will be carried out in both
countries with more patients than in France alone.
So you will need four more years,
including one year to prepare, before starting with phase III?
B: If we see some clinical benefit in
phase I/II, we might be eligible for fast track approval, because Duchenne is
an “orphan disease”, and because there is no treatment at all. But again, it is
only “if”. For a normal phase-III trial, we expect to need one hundred patients
or more and will need another four years.
So can you calculate about how long it
will take?
B: Well, we can answer in terms of best
and worst scenarios. Best scenario would be a fast track approval by 2008.
And worst scenario would mean if
something goes wrong with this or another trial for another disease, this would
be a complete stop. That is possible, too?
B: It is possible, too. You cannot rule
it out, no.
If dystrophin transfer works, at what age
should the children be treated?
W: The younger the easier it will be.
Maybe at 5 years of age, or 3 to 5 years
Will the medication be the same for all
Duchenne boys and not have to be individually designed as is expected for exon
skipping? And how often will one have to repeat the treatment?
B: Yes, the plasmids with the cDNA of the
dystrophin will be the same for all patients. W: Perhaps we will need a booster
every 6 months. So there might be an initiation dose to get to a certain level
of dystrophin and thereafter only maintenance doses.
The cDNA of the gene, that is
transported, is not integrated into a chromosome?
B: It should not. The probability is very
low. It has never been demonstrated using plasmids or viruses in intramuscular
delivery.
W: And we should get long-term
expression.
How will the treatment look? How will you
produce the pressure?
W: With a blood pressure cuff. We will
have to see how easy the procedure is. Maybe it could be done in the doctor’s
office. It will look like an infusion. The pressure is produced with a pump,
about 500 mm mercury, less than an atmosphere.
So we can have some final words, addressed
to the parents.
B: Because we are getting e-mails from
everywhere, asking the same question about the time we need, we always give the
same answer: I know it is frustrating for the parents that it takes so much
time for the clinical development of any drug, but especially for Duchenne
dystrophy. It is also frustrating for us, but we try to do it as quickly as
possible. On the other hand, we also have to follow very strict rules, because
this is for the sake of the patients, to make sure that nothing wrong happens.
So this is why it takes so long.
W: Another thing the parents should know
is that this gene transfer will not be a cure. The objective in all three
phases of the trial is to preserve hand function. We are only treating a limb,
the forearm And this is an important first step that will improve the quality
of life. When we see that this first step works, the next step will be
treatment of the legs and then possibly the respiratory muscles and the heart.
But right now the technique does not work well with heart muscles. Maybe we can
make it working better somehow at a later time. The injections are regional,
not systemic. In these first trials, we inject the plasmids into a vein at the
wrist so that they get into all the muscles from the cuff down to the end of
the hand. Again, it is not a cure but it is something to start with.
That was quite important to say. Thus
this treatment will not affect the life expectancy of the children. To achieve
this, will it take 20 to 30 years more?
W: Oh no, it will be sooner. B: Because
if you show clinical benefit with the forearm, then you can move on to other
limb muscles and even to the respiratory muscles.
W: And as more people work on the
regional technology, it will become better and better. So, please understand,
that these are important advances and that things are moving into the right
direction.
On behalf of the organizers of
this conference and certainly also in the name of the families with Duchenne
boys, I wish you all the necessary success with your research work and thank
you and your colleagues for your dedication and efforts to find an effective
treatment for Duchenne muscular dystrophy.
In the second interview, the
planned clinical trials using the transfer of very shortened cDNAs of
dystrophin genes were discussed. As an introduction, this technique is
summarized first.
In the laboratory of Professor
Jeffrey Chamberlain in Seattle, the scientists have performed considerable
engineering of the shortened dystrophin cDNAs, the combined exons of the gene.
They have identified a version that is very functional and highly effective at
combating dystrophy in the dystrophic mdx-mouse. This new ‘micro-dystrophin’
lacks most of the central portion of the dystrophin protein and the very end of
the protein. When tested in mice, this micro-dystrophin was able to almost
completely block the development of dystrophy, and was able to reverse many,
but not all features of the disease when given to adult mdx mice.
With experiments published in
the August 2004 issue of the journal Nature Medicine (10, 828-834, 2004), the
American researchers found a new method by which to transfer the
micro-dystrophin gene into the muscles of adult mice with muscular dystrophy.
This systemic delivery into the blood circulation involves inserting the
micro-dystrophin into adeno-associated virus type 6 (AAV6), a newer type of AAV
viruses that was found to be highly active at delivering genes to muscles. The
investigators injected the AAV6-micro-dystrophin into the bloodstream of mice,
together with the protein VEGF (vascular endothelial growth factor) which makes
the blood capillaries permeable for a limited time. It could then be shown that
essentially all the voluntary muscles of the mouse were now making new
dystrophin at very close to normal levels. The muscles showed an improvement in
their function, and testing the muscle breakdown by measuring serum levels of
creatine kinase showed a whole body improvement of the dystrophy. This work
shows for the first time that it is possible to deliver new dystrophin genes to
all the muscles of an adult mouse. The focus of research now will be to
determine if the method is safe and whether it can be scaled-up for larger
animals and eventually applied in the clinic.
Dr. Olivier Danos is the
Scientific Director of Généthon at Evry near Paris. He presented data on
several different serotypes of adeno-associated viruses used for therapy
studies, viruses with different surface structures. Serotypes 1 and 6 are much
better than types 2 and 5 for skeletal muscle gene transfer in mice.
Preliminary studies showed that an arterial injection of AAV containing
microdystrophin into the hind limb of a grmd dog with muscular dystrophy led to
production of low levels of dystrophin in muscles of the leg. His group is also
studying different gene regulatory elements to put into AAV vectors to control
the dystrophin production, and showed very encouraging data using a promoter of
the desmin gene.
With the support of AFM, Généthon is
currently gathering pre-clinical data on the use of AAV vectors for
micro-dystrophin gene transfer into the muscle of Duchenne patients. This work
should lead to a clinical trial within the next three years.
At the end of the first interview it was
said, the aim of the study in France is to improve hand function only, it will
not be a complete cure for Duchenne dystrophy. And if that works, one can
probably extend this method to other limbs and to the lungs. If you are using
microdystrophin for a therapy, what you will really get will be a Becker-type
disease, isn’t it?
C: We hope that, at the minimum, it would
convert the characteristics of the disease into the milder Becker form of
muscular dystrophy. It is possible that it may work better than that. From the
experiments with animals it is difficult to predict the clinical effect in
children. That is one of the reasons why we hope to get the technology into the
clinic to find out how effective it is.
D: We still do not know the mechanism of
correction with micro-dystrophin. We can only make educated guesses based on
our many years of experiments with transgenic mice. So, we have data that tell
us that it will lead to some correction. However, whether it is going to be
Becker-like, or whether it is going to be better, is impossible to say until we
have done the trial with humans. That is one reason to do localized injections
in the first trial, and then we can look at the muscle function in a human
patient and be able to understand how well this micro-dystrophin works.
But if it works, will it be a systemic
method?
D: Today we cannot tell you how the
treatment is going to be done, with what vector and by which delivery. We are
just beginning. We are discussing how we can administer the vector systemically
and get a correction in the entire musculature of a mouse. This is quite
different from the situation a year ago. I think this is progress, but it is
just the beginning of something new, and we are not there yet. The outcome of
our experiments is just totally open and there is still much more work to do.
C: There are several stages to find out,
how we can apply this technique to the patients. The first must answer the
question of how well the microdystrophins work in a human muscle compared to
the minidystrophins and the entire full-sized dystrophin. And that will likely
begin with injections into small muscles.
Will this be done in human studies in
what you call phase I?
C: Yes, that’s right. And if that is
successful, we can scale it up like it is being done in the other trial with
plasmid DNA, to try to improve the hand function. At the same time, we will
have to develop methods with animals to get a distribution of the gene to a
much larger region of the body, perhaps into an entire leg, an entire arm and
then, eventually into the whole body. But it’s going to take a lot of work to
see if that will really be possible to do first in animals and then in
patients.
D: There are a number of things that have
to be tested in clinical trials: The function of microdystrophin after it is
newly made, the best route of delivery, and also the vector itself. If
adeno-associated viruses are used, we must know which particular type would be
best. In clinical trials for a normal drug, you check for toxicity in phase I,
then you increase the dose, then you do that in more patients, that is quite
straightforward. Here, we plan local injections to look at microdystrophin
function and safety, and then we may try another mode of distribution. So,
regulatory agencies will say: this is not a phase I and then phase II, this is
a phase I and another phase I, because the conditions are different. It just
means, we are going to a complex system with our clinical trials.
Will you work together in the future on
these trials?
D: This is not decided. So far we have
been working in parallel with the same kind of ideas and tools. This workshop,
this round table, is so useful because we started to talk and are convinced it
would be much better if we would work together. Otherwise, Jeffrey in Seattle
and we in France would have completely different clinical trials.
C: It is important to have the different
groups talking to each other and to meet on a regular basis and to see where we
can help each other out. But at the same time, one does not want to completely
merge all the research efforts in the world and have only a single program
because that tends to stifle innovation and creativity. If you don’t have
independence and competition, then you may not make the next breakthrough.
D: In such a new and complex program, it
is always difficult to make decisions. Should we work with this or that
micro-dystrophin? Should we use this or that AAV serotype? Now we have several
centres going in the same direction. Each is going to make different choices
and that is good because you can never know what the results will be before you
do the experiment. If you have only one big program that goes in one direction,
the chances that something goes wrong are much higher.
And then you have to start from scratch
again.
D: Yes, and I don’t think that it would
save money in the long run. Such a large program would be much less motivating
for the people doing the work. The new way is to work in parallel, to exchange
information and to try not to duplicate efforts. Eventually this will reduce
the costs of a clinical trial.
To get at the important question the
parents always ask for instance: Will my son, who is 10 years old, still profit
from this, when he is 15 or 20? Will it come fast enough to save my son?
Therefore this question: When will you be able to start a trial, how long will
it take for each phase?
D: There are other trials, but I do not
see that any of these trials are going to save patients. They are likely to
bring us important information, but they will not save patients. Of course,
anything can happen, something we do not know today can be found and have
positive impact. We are doing the best we can with the information we have
today. It is a long process, development of a gene therapy is full of hurdles
and problems. We solve one problem, then we go on to the next. We would love to
be able to speed up the process, but it is just humanly impossible.
C: Research has to go in phases, from one
area to another. We have worked for a long time mostly with the mouse model for
muscular dystrophy, trying to understand whether it will even be possible to
have an impact on this disease. And to take that from the mouse into the
patients is a very slow and difficult process. Together with several groups
over the next two or three years, we will probably begin studies with the
microdystrophins to see if they are going to be safe and the methods of
delivery will also be safe. Then we will look at the safety of more large-scale
experiments. But these experiments with microdystrophins are only one type of
approach to cure the disease. There are also advances being made in other areas
of research. Known drugs are being studied, better steroid hormones are being
looked at, and things like this. And hopefully, they will have a very positive
impact on the disease also. All this will help the patients do better and live
longer. And hopefully, an improvement in the disease will come a little bit
faster. However, it is quite difficult to know how long it is going to take to
really have an effective treatment.
D: It is always a problem giving dates.
Anything can happen. We decided at Généthon to design an optimal pathway. This
is just to put on paper, the time lines, how long each step will take. And if
we do that, we can say, o.k., we will finish the animal studies at this time,
then move on to toxicology, and then to the clinical trial. If everything goes
perfectly well, and if there are absolutely no scientific problems arising -
but that is not going to be true - we would start a trial in two years, in
October 2006. That is about the time it takes to start one initial clinical
study with microdystrophin. We can say that, but we know that we will have to
revise these predictions all the time. So, it is by no means a firm date, and
we will not say we have to meet a deadline. That is all I can tell you about
times and how long it will take.
It was said at the meeting that the
development of a normal drug takes 15 and more years.
D: Yes, and we are still at the first
stage of the 15 years. We are really at the beginning. Obviously, we could say:
from now on it will take 15 years. But, on the other hand, we do not want to
loose the hope that something else will happen that is not gene therapy. There
could be a new drug that will totally change everything, and we have to be
ready for that.
But what can one say to the parents about
the time? That it may take 10 years or 20? Their sons will be dead by that
time. What can they do? Treat them as well as possible to keep them in good
shape if something comes along, that they can benefit from?
C: That is really the best they can do,
to take advantage of the best possible medical care and management of the
disease that we currently have available. And even without a miracle drug or
gene therapy break-through, the quality of care for the patients has improved
tremendously compared to what it was 10 years ago. So this better quality of
life of the patients is also slowly increasing their life span.
One must just take advantage of even
small improvements in medical care and of new drugs that come along. Similar
things are happening with cancer. There is no magic bullet that will cure
cancer, but there are drugs that slow down some types of cancer. And there are
many different types of treatment that can come together and have a very major
impact in improving the life of the patients. My guess is that we will see
things like that with muscular dystrophy.
Gene therapy may not be a complete cure,
but it could strengthen the muscles enough so that patients can benefit from
other approaches which will take them to an even better state of health. It is
difficult to predict how these things might work, and the best hope is to keep
as many research approaches under way as possible.
Another area is to try to avoid the birth
of some Duchenne children by better carrier detection and genetic advice, for
instance after a Duchenne boy was found by screening soon after birth. But not
only the immediate family should be counselled, but also the families that are
related to the mother. If the mother of a Duchenne boy is a carrier, her sister
can be a carrier, too. This way, one can even avoid first cases in the related
families. Carrier detection becomes more and more precise, the latest
development is the new MLPA method of the MRC company in Amsterdam.
D: That will reduce the number of
patients, sure, but will not eliminate the disease.
Because there are too many new
mutations. But if there is a new mutation, it can start a new series of
Duchenne boys in the following generations of families.
C: Screening at birth and being able to
offer genetic counselling in time is a very important approach. And these new
carrier assays are another advancement that we have seen over the last 10 to 15
years. But even with the information available, it seems to be difficult to get
that out to the patients and their families and let them know what it means.
The family doctors know the families, they should tell the immediate family
what their responsibility actually is: to warn their relatives.
C: One of the problems of this disease
is, that the gene that is defective, is such a large and very complex gene. For
a long time, it has been difficult to screen it effectively for mutations. But
the advances of DNA technology have really simplified that enormously. It is
going to be important to get the advances out of just a few research
laboratories and make it more widely available throughout the world, so that we
can have an effect on the frequency of the disease.
One of the reasons that Duchenne
dystrophy is a common genetic disease, is that it arises spontaneously and at a
higher rate than in any other known inherited disease. So it is always going to
be with us. And that is why we need to continue working as hard as we can to
develop a treatment.
Now, one other question: What
actually is the relationship between Généthon and the French association AFM?
D: Généthon is a creation of AFM. It is a
non-profit research institute whose main goal, 15 years ago, was to establish
the physical map of the genome and to identify genes associated with genetic
diseases. In 1997, I was invited to join Généthon in order to start a research
activity centred around gene therapy. At present, most of Généthon works on
gene therapy for genetic diseases. And 85% of the budget of Généthon comes from
AFM. The rest comes from government grants and other sources.
At Généthon, we live together, literally
in the same building, with the AFM. Every day, we meet, talk and have lunch
with parents and the patients. This is a very important and constant motivation
for us
The AFM is very successful in getting
money for research, that is unbelievable. --- How many people are actually
working on this gene transfer project at Généthon?
D: We have a team of about ten people who
are responsible for moving this project forward using the common resources of
Généthon. Luis Garcia and Jean Davoust who participated in this conference are
important players in this project. All together over 50 scientists and
technicians are working on projects related to muscular dystrophies at
Généthon.
And how many are working in Seattle?
C: There is core of 6 or 7 people in my
laboratory who are working on the micro-dystrophin project, but we have about
three times as many who are working on other approaches towards muscular
dystrophy.
There is another research laboratory in
Seattle, headed by Rainer Storb, in the Hutchinson research laboratories.
C:. Rainer Storb is one of the pioneers
on bone marrow transplantation, working mostly with dogs. He has now developed
a colony of Duchenne dogs. We are collaborating with him to test our
microdystrophins in dogs. But his own research group is looking more at
stem-cell based approaches using the dog to develop a treatment. Over the last
year, several researchers have moved to Seattle and begun working on muscular
dystrophy. Among them are Marie-Terese Little, Rainer Storb, and Stanley
Froehner who came from the University of North Carolina. We have now informal
collaboration between many different laboratories.
We have time for a final word to say to
the parents..
D: The parents are in a very difficult
situation. Because we are asking them to wait, this is the answer to your
initial question. They will have to wait for a long time. The first trial will
start in a couple of years. We researchers should be very humble, because we
cannot make big predictions. We are only certain about the data we have and we
know what we are going to do next. But, the parents should not lose hope, and
that is very important. We are asking for faith, and that is very difficult. Of
course, we can think about the future and carry some hope for the future. I
wish I could do more than that, but nobody can.
C: I agree with that entirely. The
important thing is to have hope, and to know that targets have been set. It is
a slow progress and it never moves as fast as one would like it to, including
us in the laboratory. But it is coming along, and, looking back 15 or 20 years,
when I first started working on muscular dystrophy, it was very difficult to
imagine that there could be an effective treatment for this disease. And now,
we can imagine that there will be a treatment for this disease. We do not know
exactly what that treatment is going to be or when it is going to come. But
what we have seen with the animals, it is possible to have a major impact on
this disease. Now we just have to struggle to find a way to make that a reality.
And we can only bring things along as fast as we can.
The last question could be, is there
enough money for this type of research? The parents themselves, when they have
a young Duchenne boy, believe, they have to collect money, so that research is
being performed to help their son. The amount of money will not be large, but
it is important, too, isn’t it?
C: Money is always important. There is a
lot more money going to muscular dystrophy research now than it was 10 years
ago, but I would never say, it is enough. There are always more things that we
can imagine doing if we had more money. There are things you could bring on
faster, but unlimited amounts of money would not bring an instant cure for this
disease. There are things that cannot be made faster.
D: We need money for all kinds of things
and we need it for the long term. But we need money also today to train young
people, and they will be the ones to make progress in 10 years from now. I
think the action of parents in collecting money is very very important, because
they are a group of people who have said, “we together are all going to get
money and give it to this project”, then it is money that is earmarked for a
certain activity and must be concentrated on this activity. So it is very
different from paying taxes, and then waiting for the government to perhaps
allocate some to Duchenne research. That is why it can be so efficient, and
this is why parent associations are so helpful. Without them, our work could
hardly exist.
Thank you very much for this interview. And
certainly also on behalf of the families, I thank you for your dedication and
wish that through your efforts a treatment for Duchenne muscular dystrophy will
be found rather in the near than in the far future.
Professor
Jerry Mendell at the Children's Research Institute of Ohio State University in
Columbus/Ohio is preparing a gene transfer trial with the aim to convert the
symptoms of Duchenne into the much milder symptoms of Becker dystrophy.
The vector used will be a
modified adeno-associated virus of serotype 2, called AAV 2.5. It will contain
a minidystrophine gene construction, D3990, whose rod regions R3 to R21 and the
C terminal end of the cDNA are removed. The dystrophin produced will be about
one third as long as the normal protein. This minigene has been developed by
Dr. Xiao Xiao of the Universtity of Pennsylvania in Pittsburgh and then used
for successful gene transfer studies in mdx mice. Dr. Jude Samulski, director of
the Gene Therapy Center at the University of North Carolina in Chapel Hill and
of the biotechnology company Asklepios will be responsible for the large-scale
preparation of the vectors to be used in the trial. This work is being
supported by the Muscular Dystrophy Association of America with an award of 1.6
million US$.
After the toxicology and bio
distribution studies with animals are completed and the permission of the
regulating agencies obtained, the trial will begin in the second half of 2005.
It will be a phase-I/II trial with 6 Duchenne patients whose mutations of the
dystrophin gene are known. They will be at least 10 years old so that they can
give their informed consent.
The injections will be done
double-blind into the biceps muscles using vector on one side and salt solution
on the other, guided by magnetic resonance imaging. Two different doses will be
used for each group of 3 patients. After 3 and 6 weeks, muscle strength will be
measured quantitatively. Also after 6 weeks, a muscle biopsy will be performed
to check for the presence of new but shortened dystrophine and of possible side
effects.
To finish this report, a
conclusion is attempted which is partly based on comments made by Professor
George Karpati in Montréal.
At this meeting, two research
gene transfer approaches were thoroughly discussed: First, the planned
administration of a microdystrophin gene construction with adeno-associated
viruses into the blood circulation of dystrophic mice and, second, the regional
administration under pressure of the combined exons of the full-length human
dystrophin gene with plasmids in the first clinical Duchenne gene therapy trial
with patients.
Microdystrophin transfer with
viruses. The first approach has shown impressive results. A single tail-vein
injection of a relatively small amount of adeno-associated virus carrying only
about one third of the dystrophin exons with a muscle specific promoter
resulted in a generous production of shortened dystrophin in all skeletal
muscles and in no other organs of the experimental animal used, the dystrophic
mouse. There was no appreciable toxicity.
These impressive results may
be explained by (1) an extraordinarily specificity of this type of viruses for
skeletal muscle fibres, (2) the use of a promoter which activated the gene
construct only in muscle cells, and (3), the simultaneous administration of
VEGF which makes the capillary blood vessels permeable for a short time.
Of course, it is not
guaranteed that all these favourable circumstances would also materialize in
children, and the therapeutic effect of the very short microdystrophin may also
produce only a mild mitigation of the severity of the Duchenne symptoms or none
at all. However, in view of the possible relative safety of the procedure,
cautious human trials appear to be justified. And major technical and financial
problems might arise when the large-scale production is attempted of the
viruses with their genetic charge in the required quality necessary for this
novel type of drug to be used in children.
The positive aspects of this
approach are in summary: (1) The preclinical studies in mice gave favourable
results, (2) the method seems to be safe, and (3), no immune reactions were
caused against the vector material or the new dystrophin.
However, there are also
uncertainties and possible negative aspects: (1) The microdystrophin might
produce only an insufficient clinical improvement, (2) the time course of any
therapeutic effect has not yet been studied, (3) studies with larger animals
than a mouse, for instance with the dystrophic dog, should be undertaken before
children are treated, (4) the large-scale manufacturing of the vector may be
prohibitively expensive, and (5), the maximal amount of the adeno-associated
viruses injected into the blood circulation of children without negative side
effects is not known.
Full-length dystrophin
transfer with plasmids. The intravenous administration of a very large number
of plasmids containing all the combined exons of the dystrophin gene, its cDNA,
with its own control elements into a leg of dystrophic mice under pressure
resulted in a variable but often appreciable production of normal dystrophin in
all the leg muscles with relatively little side effects. In the completed human
phase-I-trial, the local administration of similar "naked" genetic
material DNA proved to be safe, but the percentage of dystrophin-producing
muscle cells was never larger than 25%.
In the second phase of the
trial with Duchenne patients, it is planned to inject a rather large volume of
similar naked DNA into a vein of the forearm shortly blocked with a tourniquet.
It is expected that a high percentage of muscle cells will produce new
dystrophin and thus improve the hand function of the patients.
The positive features of the
proposed study include: (1) The feasibility of this procedure was proven in
monkeys, (2) a large amount of naked DNA can be safely introduced into human
muscles, and (3), naked DNA does not cause immune problems.
Uncertainties or even negative
aspects include: (1) For practical reasons, one tries to treat forearm muscles
first although the hand function of Duchenne patients deteriorates rather late,
(2) the large volume injected and the tourniquet application may cause
significant collateral damage, (3) the longevity of the therapeutic effect is
not known, and (4), the large-scale production of the plasmids in the required
quality will be expensive.
The future: Since the discovery of the
dystrophin gene in 1986, research for a therapy of Duchenne muscular dystrophy
has progressed considerably. In addition to the two techniques discussed at the
meeting and now in development for clinical trials, there are other promising
approaches as, for example: exon skipping, stop codon read-through, upregulation
of utrophin, and treatments with drugs like prednisone or other substances
found active in animal studies. As explained in the interviews, it will take
still many years until an effective and safe therapy, based on the two
discussed methods, will be available for the patients. Therefore, research on
all other approaches must go on without any delay. But practically all these
other techniques, as soon as they are sufficiently tested in animals, will have
to go through the different phases of clinical trials, too. Thus, it is
unlikely that any one of them will be able to produce an effective and safe
therapy for Duchenne boys faster than the transfer of the gene with plasmids or
viruses.
Participants
of the round table conference
Scientists:
Serge
Braun, Transgène, Strasbourg, France
Barry Byrne, University of
Florida, USA
Elisabeth Barton,
University of Pennsylvania, Philadelphia PA, USA
Jeffrey Chamberlain,
University of Washington, Seattle WA, USA
Jamel
Chelly, Institut Cochin, Paris, France
Giulio
Cossu, Universitá la Sapienza, Rome, Italy
Oliver
Danos, Généthon-CNRS, Evry, France
Jean
Davoust, Généthon-CNRS, Evry, France
George Dickson, Royal
Holloway University, London, UK
Luis
Garcia, Généthon-CNRS, Evry, France
George Karpati, McGill
University, Montréal, Canada
Robert Kotin, National
Institutes of Health, Bethesda MD, USA
Jerry Mendell, Ohio State
University, Columbus OH, USA
Terence Partridge,
Hammersmith Hospital, London, UK
Thomas Rando, Stanford
University, Stanford CA, USA
Lee Sweeney, University of
Pennsylvania, Philadelphia PA, USA
Sin'ichi Takeda, National
Institute of Neurosciences, Tokyo, Japan
Jon Wolff, University of
Wisconsin, Madison WI, USA
Dominic Wells, Imperial
College, London, UK
Xiao Xiao, University of
Pennsylvania, Pittsburgh PA, USA
The scientists are listed with their abbreviated addresses and without
any titles.
Most of them are
professors and all have an MD and/or a PhD.
Parents’
representatives:
Filippo
Buccella, Duchenne Parent Project, Italy
Nick
Catlin, Duchenne Parent Project, UK
Christine
Dattola, Duchenne Parent Project, France
Brian
Denger, Duchenne Parent Project, USA
Sally Hofmeister, Aktion
Benny & Co, Germany
Rod Howell, Muscular
Dystrophy Association, USA
Peter McPartland, United
Parent Project Muscular Dystrophy, UK
Luc
Pettavino, Association Monégasque contre lesMyopathies, Monaco
Christine Cryne, Muscular
Dystrophy Campaign, UK
Jenny Versnel, Muscular
Dystrophy Campaign, UK
Michel
Villaz, Association Française contre les Myopathies, France
Elizabeth Vroom, Duchenne
Parent Project, Netherlands
This report was written
by:
Guenter Scheuerbrandt, PhD
Im Talgrund 2,
D-79874 Breitnau, Germany.
e-Mail:
gscheuerbrandt@t-online.de <mailto:gscheuerbrandt@t-online.de>
A report on all research
approaches with results up to August 2003 can be seen in English, German,
French, and Spanish at http://www.duchenne-research.com. Those who wish to
receive the report on the first Monaco Round Table of 2004 amd future updates
should send their e-mail address to Günter Scheuerbrandt