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The new Harrison Ford film, Extraordinary Measures, hitting UK cinemas from 26 February, is a fictionalised account of the development of a treatment for Pompe disease, a rare genetic disorder. Pompe disease (glycogen storage disease type 2, acid maltase deficiency) is an enzyme deficiency with devastating effects – progressive muscle weakness and, in the severe infantile form, gross enlargement of the heart. Until fairly recently, the infantile form of the disease was invariably fatal within the first year of life. Now, however an effective treatment is in place.

While the increased awareness that the film’s fictional account brings is very welcome, the real story of how that treatment came about is a fascinating one (1) and laboratory animals play a starring role. The long road to a treatment started in 1932 with the first observation of the disease by Dr JC Pompe, after whom it is named. Pompe described accumulation of glycogen in muscle tissue, which was a puzzle, as the enzymes involved in the usual metabolism of glucose and glycogen were all present and correct. The solution to this puzzle had to wait until Christian de Duve’s 1974 Nobel Prize-winning discovery of lysosomes in 1955. These cellular compartments or organelles are the ‘recycling units’ of animal cells. They have an acid environment and their own specific set of enzymes for breaking down cellular components.

De Duve was carrying out ‘blue skies’ research, with no thought of direct medical application. However, as so often in research, a breakthrough in our basic understanding of biology led to medical applications. In this case, de Duve’s colleague Henri Hers realised that the deficiency of a lysosomal enzyme (alpha glucosidase) for the breakdown of glycogen would explain the symptoms of Pompe disease. This proved to be the case, and Hers established the principle of lysosomal storage diseases, of which around 40 have now been described, in 1965. Before moving on, let us note the role of laboratory animals in this breakthrough. I wrote to Professor de Duve and asked what part the use of animals had played in his work and he replied that “We would not have been able to make the discoveries we made without an extensive use of laboratory animals.”(2) - a statement confirmed by his Nobel Prize lecture.

Having discovered the basis of Pompe disease, the next milestone was to develop a treatment. This proved to be very difficult, largely due to the lack of animal models. A recurring refrain from the animal rights lobby is that if the humane use of animals in medical research was banned, scientists would soon find other ways to ensure medical progress. That comforting belief is belied by the series of attempts, some of them pretty desperate, to treat terminally ill children over the next 25 years. None of them worked.

The next great leap forward came from The Netherlands in 1990 and relied on the use of laboratory mice. Enzyme replacement therapy (ERT) had long been suggested as a potential treatment for lysosomal storage diseases but had never succeeded. In the case of Pompe disease, where large amounts of enzyme were needed in the muscle, introduced enzyme was simply soaked up by the liver. Two Dutch scientists, Arnold Reuser and Ans van der Ploeg, had the idea that phosphorylated enzyme would be taken via by the mannose-6-phosphate receptors in lysosomes, allowing the enzyme to be targeted.

However the supply of phosphorylated enzyme was small – nowhere near enough to treat a sick child. How could efficacy be demonstrated, in the absence of an animal model? In an ingenious experiment (3), they used specific monoclonal antibodies to demonstrate that when bovine phosphorylated alpha glucosidase was introduced to mice, it was taken up by heart and skeletal muscle lysosomes and caused a significant increase in enzyme activity – a 43% increase in skeletal muscle and 70% in the heart. An increase that, if repeated in humans, would result in the level of enzyme found in the healthy population. With characteristic understatement, van der Ploeg et al concluded "...we think that the original idea of enzyme replacement therapy for treatment of lysosomal storage diseases deserves new attention." At last, thanks to this ground-breaking work, a treatment for Pompe disease was a real possibility.

Now that there had been ‘proof of principle’ all that was needed was for a pharmaceutical company to spend millions of dollars in developing a treatment. Understandably perhaps, given the rarity of the disease and the inability to demonstrate actual efficacy, there was no immediate rush. Fortunately, at this point two animal models became available that allowed scientists to demonstrate that not only did the phosphorylated alpha glucosidase make its way to the lysosomes, it also had a beneficial effect.

From 1998 onwards, transgenic mice with Pompe disease, developed in Rotterdam and elsewhere, were used to demonstrated the efficacy of alpha-glucosidase enzyme. At the same time the potential of ERT was also illustrated, more dramatically perhaps, by YT Chen at Duke University, using quail. The quail had the same enzyme deficiency as found in humans, resulting in muscle weakness. After injection with the enzyme, they recovered to the extent of one subject actually flying around the lab (4). The evidence was therefore now pretty convincing – it was time for human trials.

The big problem was in producing enough enzyme for humans, even for babies. This required substantial industry investment. Two rival approaches were tried. A Dutch company, Pharming, produced the enzyme in the milk of transgenic animals for use in a trial led by Ans van der Ploeg, whose PhD research had led to the original breakthrough. The transgenic animal used was the rabbit, on the grounds that a human alpha-glucosidase-producing line could be established quite quickly. This work was used in a successful clinical trial, the results of which were published in The Lancet in July 2000 (5).

Another trail was carried out by YT Chen, using enzyme produced via Chinese Hamster Ovary (CHO) cell culture, by Synpac, a Taiwan-based company. This trial was also successful.

What follows next is a slightly convoluted story. The short version is that a third company, Genzyme, with an existing enzyme replacement therapy for Gaucher disease, bought out both Pharming and Synpac. In the end, they didn’t use either of the enzymes produced by these companies but developed their own, in-house CHO product, now marketed as Myozyme. This was a difficult decision – how could they decide which of the competing products should be invested in to produce a commercial treatment? The answer was what Genzyme called “The mother of all experiments” which compared the different products in transgenic Pompe mice. The result led to the availability of the treatment we have today.

However, the eventual production system is a technicality that need not concern today’s patients. Their concern is that an untreatable, terminal illness is now treatable. If you go and see Extraordinary Measures do bear in mind the starring role that doesn’t appear in the cast list – that of the mice and quail that made this treatment possible.

Kevin O’Donnell

Edinburgh

1. http://pompestory.blogspot.com
2. Letter from Christian de Duve to Kevin O’Donnell, 4 March 1997
3. Intravenous Administration of Phosphorylated Acid Alpha-Glucosidase Leads to Uptake of Enzyme in Heart and Skeletal Muscle of Mice http://www.jci.org/articles/view/115025
4. Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail http://www.jci.org/articles/view/1722/pdf
5. Recombinant human alpha-glucosidase from rabbit milk in Pompe patients http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(00)02533-2/fulltext#article_upsell (free registration required)

Biographical note.

I should declare an interest. I am a professional scientist, however my involvement with Pompe disease dates from the diagnosis of our first child, Calum, with infantile Pompe disease in 1993. At that time the disease was still untreatable and Calum died at 8 months of age. Following that I had the great privilege of participating in an international community of patients and scientists that championed the development of a treatment for Pompe disease. They don’t appear in the cast list of the film either, or the book on which it is based The Cure by Geeta Anand. This prompted me to write the real story down – I think it’s a better story than either rthe book or the film though not, sadly, as well written. Your comments welcome at http://pompestory.blogspot.com You can find out more about Pompe disease from the following sites:

International Pompe Association www.worldpompe.org
Acid Maltase Deficiency Association www.amda-pompe.org
UK Pompe Group www.pompe.org.uk
Genzyme www.pompe.com

Every time your heart beats it pumps blood through the pulmonary artery and into your lungs where it soaks up oxygen before bring returned via the pulmonary vein to the heart, where the next beat pumps it out through the aorta and on to provide oxygen to all the tissues of your body. All this is of course welcome to those of us who enjoy being alive, but there was one time in your life when such pulmonary circulation was a potential threat. During fetal development the lungs are bathed in amniotic fluid and the body is supplied with oxygen via the placenta, and at this stage the pressure of blood being circulated through them would damage the delicate fetal lungs. Fortunately evolution has provided mammals with a means of avoiding this damage, a blood vessel called the ductus arteriosus which connects the pulmonary artery to the aorta, allowing most of the circulating blood to bypass the lungs. As a baby takes its first breaths after birth the ductus arteriosus begins to close to allow normal pulmonary circulation to take place, a process that is normally complete within a few days. Unfortunately the ductus arteriosus does not always close, causing a condition known as Patent Ductus Arteriosus (PDA). If left untreated PDA can cause breathing difficulties and eventually lead to congestive heart failure, and is a particularly common and serious condition for preterm infants. While it sometimes resolves itself with minimal intervention in many cases surgery is required to correct the fault. Now research on mice has enabled scientists to uncover a key process in the closure of the ductus arteriosus that may show the way to less invasive treatments (1).

The team lead by Dr Steffen Massberg and Dr Katrin Echtler in Munich knew from previous research that the closing of the ductus arteriosus was associated with the release of cytokines that are usually associated with the inflammatory response seen when tissue is damaged. Knowing that such inflammatory responses recruit platelets, irregularly-shaped bodies produced by bone marrow cells that are crucial to blood clotting, to the damaged tissue they investigated the role of platelets in the closing of the ductus arteriosus. They decided to study the process in mice because the availability of a variety of tissue staining and genetic modification techniques which would allow them to study the whole process in great detail. They observed that within an hour of birth cells lining the ductus arteriosus were detached to provide attachment sited for platelets, and the platelets themselves quickly accumulated and soon formed a plug that stopped blood flow. They next used monoclonal antibodies and GM modification to remove two proteins that are required for platelet adhesion and activation, and found that the ductus arteriosus failed to close in the newborn mice, leading to the same problems seen in human babies with PDA. The role of platelets was not confined to the initial blocking of the ductus arteriosus, Dr Echtler and her colleagues found that the platelets also attracted the specialized precursor cells that are required to remodel the ductus arteriosus and replace the temporary plug with a more permanent closure.

So it appears that platelets play a key role in the closure of the ductus arterious in mice, but what about humans? While they could not study the process in the same detail in human babes as in mice they were able to obtain good evidence supporting a vital role for platelets in humans too. First they examined samples of ductus arteriosus taken from newborn infants who had undergone heart surgery, and observed the same modifications to the lining of the ductus arteriosus and platelet accumulation that they had seen in the mice. They then studied a group of 123 premature infants and found a strong association between low platelet counts and PDA , further evidence that platelets are required for closure of the ductus arteriosus in humans just as they are in mice.

In an interview for the BBC stated that "It is conceivable that transfusion of platelets reduces the risk of ductus arteriosus patency (lack of closure) in preterm newborns with low platelet count.". We hope that he is right and that this discovery leads to a revolution in the treatment of PDA. As for the question of where the platelets for such treatment will come from, that part is all up to you.

Regards

Paul Browne

1) Echtler K. Et al "Platelets contribute to postnatal occlusion of the ductus arteriosus" Nature Medicine Published online: 6 December 2009 | doi:10.1038/nm.2060

Hot on the heels of last weeks report of the successful use of gene therapy to treat the eye disease Leber’s congenital amaurosis comes a report that scientists lead by Nathalie Cartier and Patrick Aubourg of the French National Institute for Health and Medical Research have combined gene therapy and stem cell medicine to successfully treat two boys with the disease cerebral X-linked adrenoleukodystrophy (X-ALD).

X-ALD is caused by mutations in the ABCD1 gene that plays a key role in the transport of fatty acids within cells, and lack of ABCD1 causes long-chain fatty acids to build up within cells known as microglia and oligodendrocytes in the brain. Affected microglial cells and oligodendrocytes eventually cease to maintain the insulating myelin sheath that is required for effective transmission of electrical impolses along nerve cells, leading to brain damage and ultimately death at an early age. The disease was made famous by the film "Lorenzo's oil" which describes a dietary supplement that may delay the progression of the disease, though the only treatment that is currently considered truly effective is allogeneic hematopoietic cell transplantation where healthy bome marrow stem cells from a donor are transplanted into the X-ALD patient. Allogeneic hematopoietic cell transplantation works because the microglial gells and oligodendrocytes develop from cells that migrate to the brain from the bone marrow, so that healthy cells from the donor eventually replace some of the patient's ABCD1 deficient cells and help maintain the myeline sheath. Unfortunately it is often difficult to identify a suitable donor, and even if one is found the procedure is risky due to problems such as graft-versus-host disease where immune cells in the donated bone marrow mount an immune response against the patient's tissues.

Dr. Cartier and colleagues examined the possibility of using gene therapy to modify the patient's own hematopoietic stem cells so that they express a functioning ABCD1 gene and then injecting these cells into the patient to replace their faulty bone marrow hematopoietic cells, thereby avoiding the problem of donor and host incompatability. Rather than attempt to genetically modify and transplant all types of human bone marrow stem cells they concentrated on a subset of cells called the CD34+ cells that give rise to many cells of the immune system. These have the great advantage that they can be isolated from the blood, avoiding the need for surgery to harvest bone marrow. To assess whether genetically modified CD34+ cells could develop into cells of the immune system when injected into the bone marrow they selected the NOD/SCID mouse that lacks a functioning immune system and is often used to assess human stem cell transplantation techniques and to study aspects of the human immune system. Initial results with retroviral vectors were disappointing but using the NOD/SCID mouse model they developed a lentiviral vector based on HIV-1 that enables the functioning ABCD1 gene to safely incorporate into the genome of a significant proportion of the cells and drive ABCD1 expression in immune system cells derived from them (1). What is more they found that as well as the expected range of immune cells the genetically modified CD34+ cells migrated to the brain and differentiated into microglial cells (2). Of course if the therapy is to prevent disease progression the vector needs not only to drive expression of ABCD1 but to do so reliably for many years,. To assess whether the lentiviral vector could do this they transplanted Sca-1+ cells, the mouse equivalent of human CD34+ cells, containing the ABCD1 expressing lentiviral vector into mice that lacked a functional ABCD1 gene, and found that even 12 months after transplantation almost a quarter of microglial cells in the mouse brain expressed ABCD1 (3).

These promising results in mice were enough to persuade Dr. Cartier and her colleagues that this therapy should proceed to a pilot study in human patients who are in the early stages of this desease. While it will take several years of observation and clinical trails involving larger numbers of patients before we can be sure that this therapy is a success, this exciting news is yet another sign that gene therapy is finally coming of age.

Regards

Paul Browne

1) Benhamida S. et al "Transduced CD34+ cells from adrenoleukodystrophy patients with HIV-derived vector mediate long-term engraftment of NOD/SCID mice." Mol Ther. Volume 7(3), pages 317-324 (2003) PubMed: 12668127

2) Asheuer M. et al. "Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein" Proc Natl Acad Sci U S A. Volume 101(10), pages 3557–3562 (2004) PubMed Central: PMC373501

3) Cartier N. et al. "Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy" Science Volume 326(5954), pages 818 - 823 DOI: 10.1126/science.1171242

Back in August, Dario wrote about how basic science contributes to medical advances and today the Nobel Assembly chose to recognize the importance of such work by awarding the The Nobel Prize in Physiology or Medicine 2009 to Dr. Elizabeth H. Blackburn, Professor Carol W. Greider and Professor Jack W. Szostak for their discovery of "how chromosomes are protected by telomeres and the enzyme telomerase".

The discovery was made when Dr. Blackburn was sequencing the DNA of the tiny unicellular fresh water animal Tetrahymena, a popular model organism among scientists studying the process of cell division and the architecture of the organelles within the cell, and found repetitive CCCCAA sequences at the ends of their chromosomes. She then teamed up with Prof. Szostak who was then studying the stability of DNA molecules called minichromosomes in yeast, and as the Nobel Prize press release tells us they performed...

... an experiment that would cross the boundaries between very distant species . From the DNA of Tetrahymena, Blackburn isolated the CCCCAA sequence. Szostak coupled it to the minichromosomes and put them back into yeast cells. The results, which were published in 1982, were striking – the telomere DNA sequence protected the minichromosomes from degradation.

Professor Greider, then a graduate student working in Dr. Blackburn's lab, then identified telomerase, the enzyme responsible for making and maintaining the telomeres. This was a very significant discovery as it proved that telomeres were made using a different mechanism than that used to make the rest of the DNA sequence in a chromosome. Further research by Blackburn, Greider and Szostak and others demonstrated that telomeres are present in a wide range of plants and animals, organisms that diverged from a common ancestor over a billion years ago, and discovered that they play an important role in cellular ageing, first in yeast and subsequently in humans. Telomeres also appear to have a role in cancer, as it appears that in some (but not all) cancers elevated levels of telomerase activity contribute to the cancer cells continuing to divide and produce new cancer cells long after healthy cells would have lost the ability to divide due to the loss of their telomeres, consequently the field of telomerase inhibitors has seen intense activity as scientists seek new cancer treatments. Many of the recent insights into the role of telomeres and telomerase in ageing, inherited diseases and cancer have come from research on mice, particularly genetically modified mice with altered telomerase activity levels, and Professor Greider in particular has been at the forefront of this work for over a decade (1).

We offer our congratulations to this years' Nobel Laureates in Physiology or Medicine, their work is a striking example of how basic, curiosity-driven research on species that may appear only distantly related to us can illuminate human biology and open up whole new fields of medical research.

It did not escape our notice that the same trio who won this years' Nobel Prize were awarded the Albert Lasker Basic Medical Research Award in 2006, for the same work. We were delighted to learn a couple of weeks ago that this years Albert Lasker Basic Medical Research Award has been awarded to Professor John Gurdon and Professor Shinya Yamanaka "For discoveries concerning nuclear reprogramming". The Lasker Foundation has published an excellent summary of their contributions to this exciting field which demonstrates the absolutely crucial contribution of animal research to their work.

A couple of years ago I discussed Professor Yamanaka's work on the Pro-Test blog, and since then I have had the opportunity to discuss cellular reprogramming on Speaking of Research, which makes it all the more gratifying to see his work recognized in this way so soon.

We offer both Professor Gurdon and Professor Yamanaka our heartiest congratulations, the smart money has to be on them being summoned to Stockholm in the not too distant future.

Regards

Paul Browne

1) Blackburn E.H., Greider C.W., and Szostak J.W."Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging." Nat Med. Volume 12(10), Pages1133-1138 (2006) http://dx.doi.org/10.1038/nm1006-1133

An interesting item in the news today about research on repairing the damage to the heart caused by a heart attack. The report in PNAS can be read by those with a subscription at:

While there have been several attempts to bioengineer cardiac tissue for transplant in vitro using starting from cells seeded onto a scaffold, so far these efforts have been hampered by difficulties in getting the capillaries necessary to supply blood to enable the muscles in the tissue patch to grow properly. Due to these difficulties engrafted heart patches have until now had limited benefits on heart function in animal models of heart attack, and consequently this approach has not yet been assessed in human clinical trials.

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In this project the scientists at Ben-Gurion University started with a similar approach to that used previously by other scientists. They grew the patch of tissue from neonatal rat heart cells which were seeded in scaffolds designed to allow cardiac cell organization and blood vessel penetration after transplantation, and supplemented them with a mixture of growth factors that encourage cell survival and blood vessel growth. After the cells had been cultured in vitro for 24 hours to allow initial organization of the cells within the scaffold they introduced a new step, implanting the patch into the rat omentum, an abdominal tissue that is particularly rich in blood vessels, in the hope that the interaction with the blood vessels of the omentum would encourage the development of mature blood vessels in the heart patch.

They observed that the blood vessels of the omentum connected with those developing in the heart patch, encouraging blood vessel development and growth of cardiac muscle. The real test came when they compared the ability of omentum-grown heart patches to repair tissue damage in rats which had undergone experimentally incuced heart attacks 7 days earlier, with that of heart patches that had been grown in vitro. The result was clear, the omentum-grown heart patches had better blood vessel and muscle quality than the in-vitro grown patches and integrated more strongly into the heart. When they examined several parameters of heart function they found that the hearts of those rats which had received omentum-grown patches worked better than those of control rats and those which had received in-vitro grown patches.

So what does this mean for the treatment of heart attacks? The authors point out this is a relatively straightforward procedure that could be assessed in human trials, but also caution that the extra surgery required to grow the heart patch on the omentum would be to risky for many elderly or ill patients so it is a procedure suitable for only a minority of heart attack patients. What the authors suggest is the development of in vitro bioengineering techniques that mimic the influence of the omentum on the growth of blood vessels and muscle within the heart patch, and with this study they have begun to determine what the requirements of such in vitro systems are.

Needless to say as such in vitro techniques for stimulating heart tissue growth are developed they will need to be assessed in animal models of heart injury before they can enter clinical trials in human patients.

Regards

Dr. Paul Browne