101nd ENMC International Workshop

Therapeutic Possibilities in Duchenne Muscular Dystrophy

101st International Workshop of the European Neuromuscular Center

To celebrate one hundred international workshops on neuromuscular diseases, a special centennial workshop was held from 30 November to 2 December 2001 in Naarden in the Netherlands. A summary for scientists on the presentations and discussions of 25 invited participants has been published in the May 2002 issue of the journal Neuromuscular Disorders, volume 12, pages 421-431. However, people without training in modern biology, especially parents of boys with Duchenne muscular dystrophy and their pediatricians and family doctors, are also highly interested in the present state of research for a therapy of this still incurable disease. The following report, based on the scientific summary, is written for them and dedicated to all of the about half a million Duchenne boys of the world.

The participants reported on their own work and, in some cases, reviewed the state of research in their special field. In the following paragraphs, the presentations and discussions are summarized with as few scientific words as possible. As not all of these words could be avoided, many are explained immediately after they are used, but those which are used more often, are explained either in the introduction in the context of the basic scientific facts or in the addendum which also shows an example of how a point mutation in the dystrophin gene leads to a premature stop codon.

The complete texts of the scientific presentations are published in the October 2002 issue of the journal Neuromuscular Disorders.

Introduction

Some basic scientific facts are explained, what genes are, how they work, why dystrophin is important, and how mutations cause Duchenne dystrophy.

Genes are functional units of the genetic material desoxyribonucleic acid, DNA, in the chromosomes inside the nucleus of each cell. Its structure looks like an intertwined ladder, the double helix. The two backbones or strands of the ladder are chains composed of phosphoric acid and desoxyribose, a kind of sugar. The rungs consist of four different small molecules, the bases: adenine, guanine, thymine, and cytosine (abbreviated A, G, T, C), two of which always face each other in one rung of the double helix. For spatial reasons, the rungs can only contain the base pairs A-T and G-C. Therefore, the sequence of the bases on one strand is always complementary to the sequence on the other, as shown in lines A and B and also E and F of the example in the addendum.

The unit of the genetic information, a genetic letter, is the nucleotide, consisting of one of the bases attached to one desoxyribose which also carries a phosphoric acid group. Each of the 100 billion human body cells contains in its nucleus 46 chromosomes with genetic material totalling more than six billion base pairs, grouped in about 35,000 genes. The DNA double helix has a diameter of two nanometers, millionth of a millimeter, and its total length is two meters packed densely in the cell nucleus which has a diameter of only about 3 to 10 micrometers, thousendth of a millimeter.

Most of the genes carry the instructions for the construction of proteins, which consist of strings of amino acids, its building blocks, of which there are 20 different kinds. In the cell nucleus, the genetic instructions are copied, transcribed, from the genes to another genetic substance, the pre-messenger ribonucleic acid, pre-mRNA. Most genes consist of active, exons, and inactive sections, introns, which are often very much longer than the exons. After transcription, the introns are removed from the premessenger RNA, and the transcribed exons spliced together to the messenger RNA, mRNA, which then moves out of the nucleus to the ribosomes, the protein synthesizing structures in the cytoplasma of the cell. The ribonucleic acids, RNAs, use the base U, uracil, instead of the very similar base T of the DNA, and their sugar unit, ribose, has one oxygen more than the desoxyribose of DNA. In the messenger RNA, three consecutive genetic letters, nucleotides, signify one of the 20 different amino acids according to a genetic dictionary, the universal genetic code, which is used by all life on earth. Three of these code words are stop signs, where the protein synthesis is terminated. Thus, the genetic script uses only four letters, and its words, the codons, are always three letters long. There are no spaces between the words. In the ribosomes, catalytic acting RNAs, ribozymes, use the genetic instructions of the messenger RNA to construct specific proteins out of amino acids which are delivered to the ribosomes by another kind of RNA, the transfer RNAs or tRNAs.

Duchenne muscular dystrophy is caused by a mutation or damage of the dystrophin gene. With 2.6 million base pairs, it is the longest human gene: if stretched out, it is 0.88 mm long. Only 0,5% of the base pairs, 13,973, belong to the 79 exons of the gene, that contain the active coding sequence, the instructions for the synthesis of dystrophin, a long rod-shaped protein consisting of 3,685 amino acids. It is 125 nanometers long; i.e. if 8,000 dystrophin molecules were put together in a straight line, their overall length would be just one millimeter. Dystrophin is important for the mechanical stability of the cell membrane during muscle contraction, it is located on the inside of the muscle cell membranes to which it is anchored through a complex of many other proteins, the dystrophin-glycoprotein complex.

There are three types of mutations of the dystrophin gene: deletions, if parts of the gene are missing, duplications, if parts of the gene are repeated, and point mutations, if single genetic letters are exchanged, eliminated or added. As the reading mechanism in the ribosomes always reads code words of three letters one after the other without interruption, a mutation does not disturb the normal reading frame if the number of letters missing or added can be divided by three without a remainder. In this case, the reading frame remains in-frame and the dystrophin made is longer or shorter. If this change only concerns non-essential structures of the dystrophin, it can still be partly functional. Then, the benign form of dystrophy, Becker muscular dystrophy, develops. If the mutation shifts the reading frame by one or two letters, the reading frame becomes out-of-frame and, in most cases, a number of incorrect amino acids is incorporated into the protein starting at the mutation site until finally a new and premature stop codon is reached which terminates the synthesis. The incomplete dystrophin cannot fulfill its normal function, it is degraded and Duchenne muscular dystrophy develops. An example of a point mutation leading to a premature stop codon in the dystrophin gene is given in the addendum.

Transfer of a new dystrophin gene

With gene therapy experiments, either new dystrophin genes are introduced into muscle fibers by cell transplantation, or only the coding parts of the full-length or shortened dystrophin genes are transferred with viruses. Immune reactions against the new protein must be avoided. The original dystrophin gene is not removed, the transferred genetic material does not enter the chromosomes.

Stem cells among myoblasts: Terry Partridge (London) discussed the role of myoblasts or satellite cells which have commonly been regarded as the only cells which can form new muscle tissue and thus repair damaged muscle fibers or produce new ones. It seems now that cells which circulate in the blood can also contribute to the repair of muscle damage, but not to a significant extent. The myoblast transfer experiments 12 years ago have shown that only about 1% of the transplanted cells survived in the diseased muscle to form muscle fibers with dystrophin. These few surviving myoblasts seemed to behave like rare stem cells because each of them could form thousands of nuclei in new muscle fibers. Experiments are now being undertaken to find out what these rare active cells really are and how they can be separated from the less effective ones. There seem to be signals in the muscle tissue itself which makes them multiply, and these signals must be special substances which have to be identified. If these stem-cell-like myoblasts and the activating signals in the muscle can be isolated, myoblast transfer could probably be made more effective.

Stem cells from bone marrow: Fulvio Mavilio (Modena) reported on experiments with stem cells from the blood building bone marrow which are known to lead to normal muscle fibers when injected into the regenerating muscles of experimental animals like the mdx mouse. The transplantation of bone marrow from healthy mice, which is a mixture of many different cells, into these dystrophic mice, however, led only to very few, less than 1%, of fibers containing dystrophin. This means that the transformation of the blood building stem cells into cells which can make new muscle cells requires signals, special substances which the dystrophic muscle is apparently unable to provide in sufficient amount but which are still not well known. They may be the same as those which activate the stem cells among myoblasts. Before these cell transfer techniques can be further developed and possibly applied in children, the researchers must first understand these rather complex relationships.

Gene transfer experiments: Dominic Wells (London) gave an overview of the different gene transfer experiments which were mostly done with mdx mice. However, the muscular dystrophy of these mice is different from the Duchenne dystrophy of children, so it cannot be expected that the results obtained with mice will be the same when applied to Duchenne boys. Some of these experiments were done with transgenic mdx mice, which have an undamaged dystrophin gene inserted into their chromosomes. In these mice, the disease, could be prevented before they were born. However, the aim of research is finding a therapy to cure the disease of a Duchenne boy after he is born or when he is already several years old. As it is impossible to perform most of the necessary experiments with children, working with dystrophic animals like mice and dogs is almost the only way to develop a gene therapy for Duchenne boys. In some of the more recent animal experiments, possible treatments of the disease have been tested and the results are promising.

Immune reactions after gene transfer: Other subjects discussed by Dr. Wells were the immunological problems which are expected when, after a successful gene transfer, the protein dystrophin is made for the first time in the muscles of a Duchenne boy. This new dystrophin can be regarded by the immune system as a foreign substance which must be attacked. And the proteins of the virus used for the transport of the dystrophin gene can also provoke an immune defense. Studies in the mdx mouse have shown that such immune reactions are not generated against newly made mouse dystrophin, despite the absence of normal dystrophin in the muscles of these mice. The reason might be that there are other forms of dystrophin in other tissues which causes the immune system to tolerate the new dystrophin in the muscle. These results suggest that immune reactions may not be a particular problem in some Duchenne patients. Experiments are now being performed to determine which of the other dystrophins must be present so that the immune system does not reject new muscletype dystrophin after gene transfer.

Avoiding immune reactions: Jeff Chamberlain (Seattle), described several methods to transport the dystrophin gene into dystrophic muscles without causing an immune reaction against the newly made protein dystrophin. Adenoviruses whithout any of their own genes, gutted viruses, were used as vectors which are large enough to carry all the active genetic sequences, the complete cDNA, for the full-length dystrophin and in addition also the promoter for the enzyme creatine kinase. Promotors are DNA sequences, mostly at the beginning of a gene, that act as switches to turn a gene on or off. As creatine kinase is needed in muscle cells for the storage of chemical energy, its promoter turns the gene on only inside muscle cells. Combining this promoter sequence with the dystrophin gene makes certain that the new protein dystrophin is also made only in muscle cells and nowhere else. It is then also not made in those cells which are responsible for an immune reaction. However, because the dystrophins of mice and humans are slightly different, a mild immune reaction developed when mdx mice made human dystrophin after they received the human gene by gene transfer.

Gene transfer with adeno-associated virus: Dr. Chamberlain also discussed another vector, the adeno-associated virus, that can transport genes slightly less effectively than the adenovirus, but has the advantage of being apparently safer and longer lasting. It is much smaller and thus can only transport genetic material that is much shorter than the entire cDNA of dystrophin. To use this vector, it was necessary to determine whether shorter versions of dystrophin could also function as effectively as the full-length dystrophin to avoid Duchenne dystrophy. A large number of genes for shorter dystrophins, mini- and micro-dystrophins, were made, and then transgenic mdx mice were raised which had these shorter genes in their chromosomes, or which received them in gene transfer experiments with adeno-associated viruses. Several of the smaller dystrophins could protect the mice from their dystrophic symptoms. Transfer of the the most effective of these short genes led to a dystrophin protein which had practically the same properties as the unaltered protein: it was stable for a long time and reversed dramatically the dystrophic symptoms of the mice. These results show that it will be possible to use different vectors for a future gene therapy, and that significantly smaller dystrophin genes than the normal one can be made and transferred which may be even more effective than the naturally shortened dystrophin gens of patients with Becker dystrophy.

Experiments to make gene transfer more effective: The research team of Hanns Lochmüller (Munich) is trying to make the gene transfer with adenoviruses more effective. Muscle fibers have special protein structures, receptors, on the outside of their membranes to which the viruses attach themselves before they enter the cell. There are only very few of these receptors on fully developed, adult muscle fibers, and that is the reason why the gene transfer with adenoviruses does not work as well as it should. To improve that situation, transgenic mice were made with many more of these receptors, which increased the efficiency of the gene transfer by up to 10 times. The greater number of receptors means also that fewer viruses would be needed and thus any side effects caused by the viruses can be reduced. However, as the techniques to create transgenic mice cannot be used in humans, drugs which would increase the number of the receptors on the muscle membranes of a Duchenne boy would be needed and they are not yet known. Therefore, the researchers are now trying to change the outside structure of the viruses so that they attach themselves not to their normal receptors but to other surface structures of the muscle cell membrane.

Transfer of short dystrophins: In George Dickson’s (London) laboratory, micro dystrophins were also tested for their use for gene transfer with adeno-associated viruses and also with another type of vector. This new type was a combination of an adenovirus with a retrovirus, which, in contrast to the other viral vectors, inserts itself and the dystrophin gene into one of the chromosomes in the cell nucleus. With this new virus, gene transfer into the muscle of mdx mice was quite effective, and there was an indication that the few stem cells in muscle were transformed into satellite cells which contributed to muscle cell repair. The transferred micro dystrophin was maintained for a long time in the muscle fibers because their degeneration was reduced considerably. These results are important because a future gene therapy should ideally need only to be applied once or very few times during the probably significantly extended life time of a Duchenne boy.

Repairing the dystrophin gene

Mutations in the dystrophin gene on the X chromosome are repaired with oligonucleotides. Entire exons of the gene are removed from the mRNA to restore the disturbed reading frame.

Gene repair with oligonucleotides: Thomas Rando (Stanford) started his presentation with an overview of at least six different methods not for transferring a new gene but for repairing the damaged gene where it is located, on the X chromosome. In all of these methods, different kinds of short sequences of nucleic acids, DNA or RNA, or chimeric molecules made up of both DNA and RNA, called oligonucleotides play the decisive role. Because the sequenes of these oligonucleotides are similar to sequences of the dystrophin gene, they can attach themselves to the gene or its messenger RNA, and then can modify the mutation and allow the production of functional dystrophin protein. In Dr. Rando’s laboratory, chimeric RNA-DNA oligonucleotides were injected in vitro into myoblasts from mdx mice to correct the point mutation of their dystrophin gene. The sequence of these structures was made in such a way that they were capable of attaching themselves to the DNA structure around the area of the mutation. Up to 15% of the normal level of dystrophin was then found in the mdx myotubes, the next stage of muscle development. This technique also worked in living mice, but the amount of newly made dystrophin was lower. As most of the Duchenne boys have gene deletions and only a relative small number have point mutations, this technique will now be modified so that it can be applied to restore an out-offrame information back into in-frame after a deletion. This could be achieved if in the messenger RNA whole exons could be eliminated or single nucleotides be added or deleted. However, there are still many problems to solve before this chimera-technique could become a general Duchenne therapy.

Gene repair with short gene sequences: Robert Kapsa (Melbourne) reported on his experiments with a technique called short fragment homologous replacement in which an artificially made short gene segment replaces the corresponding one around the mutation site, so that single nucleotides in the gene are added or removed. Such an addition or deletion, if applied directly behind the site of a mutation, could restore the disturbed reading frame caused by that mutation, and preliminary experiments indicate that this is indeed possible with this technique. It would also be possible to change the borders between exons and introns, again with the aim of restoring the reading frame. In the mdx mouse, an artificially made DNA strand, consisting of 603 nucleotides with the correct sequence around the point mutation site, restored the dystrophin gene mutation in up to 20% of isolated myoblasts in mdx mice. However, when this same piece of corrective DNA was injected directly into the muscles of living mice, the gene was repaired only in less than 1% of the muscle fibers. Thus, a possible Duchenne therapy with this approach would be more promising if used as ex vivo gene conversion, i.e., myoblasts would have to be isolated from a patient, treated with the corrective DNA in the laboratory, and then the corrected myoblasts re-injected into the blood stream or the dystrophic muscles otherwise remodelled. For older patients, it would be more advantageous to use stem cells from tissues other than muscle and to transform them with this technique. However, preliminary experiments with bone marrow cells injected into mdx mice were not very successful in remodelling the dystrophic muscle. Therefore, further studies are necessary to identify and to isolate stem cells from bone marrow and other tissues whose dystrophin gene could be corrected in this way. In a possible human application, the aim would be to inject these treated cells into the blood stream of a Duchenne patient, from whom they were originally extracted. Such remodeled cells should ideally find their way into the diseased muscles and regenerate healthy muscle fibers without being rejected by the immune system.

Exon skipping: Judith van Deutekom (Leiden) described experiments with specially designed short stretches of RNA as possible drugs to repair a mutation in the dystrophin gene. If one or more exons of the dystrophin gene are missing, the reading mechanism of the genetic message can go out-of-frame. However, there are some exons, whose deletion does not disturb the reading frame. In this case, the instructions remain in-frame, but the dystrophin is shorter than normal. In many cases, this shorter protein does not protect the muscle membrane completely, but sufficiently enough to lead to the milder Becker-type muscular dystrophy. In order to change the out-of-frame information back into in-frame again, the researchers try to eliminate specific exons directly preceding or following the deleted one and thus changing the dystrophy of a Duchenne boy into the much slower Becker form. To achieve this goal, specific short sequences of RNA, oligoribonucleotides, were made. If they have an antisense structure which allows them to attach themselves to a special place inside one of these exons of the pre-messenger RNA, that particular exon will be skipped, will not be present in the messenger RNA. Therefore, its information will not be used and the reading frame will be restored allowing dystrophin to be made by the muscle cell. To show that this technique works, Dr. van Deutekom’s team was able to cause the skipping of exon 46 in muscle cells from two Duchenne boys who had a deletion of exon 45 in their dystrophin gene. This experiment was done in vitro, in the laboratory on muscle cells obtained through a biopsy from the two boys. In at least 80% of the treated muscle cells, new but slightly shortened dystrophin was present. As the information from both exons, 45 and 46, could not be used, 108 of the 3,685 amino acids of dystrophin were missing. This technique was also applied for the skipping in vitro of 14 other exons, 2, 19, 29, 40, 41, 42, 43, 44, 45, 49, 50, 51, 52 and 53 to restore other out-of-frame mutations, and it is being tried in cases with duplications and point mutations, which also can cause out-of-frame genetic information. The next step then was to find out whether exon 46 could be skipped in vivo, in a living organism. Thus muscles of mice were injected with the special antisense RNA oligonucleotide that causes the skipping of exon 46. First results showed that exon 46 was removed in about 10% of the messenger RNA in the injected muscles. It is known that deletion of exon 45 in boys leads to Duchenne dystrophy while the combined deletion of exons 45 and 46 causes Becker dystrophy. Therefore, it is to be expected that if this technique would also work in Duchenne boys, their dystrophy could be changed into Becker dystrophy. Studies are being undertaken to improve the method, to modify it so that the oligonucleotides could be injected into the blood stream, and finally to perform clinical studies with Duchenne patients. Up to 65% of the boys with deletions, or about 40% of all Duchenne boys, could possibly be treated by skipping one of the 15 exons mentioned above.

Stabilization of oligonucleotides: In his second presentation, George Dickson (London) reported on work with an antisense oligoribonucleotide whose ribose units of its backbone had each an additional group of one carbon and three hydrogen atoms attached. This made this potential drug much more stable. If its structure is chosen so that it combined to the special base sequence which directs the splicing of the pre-messenger RNA, specific exons can be skipped, deleted, from the messenger RNA. With this technique, exon 23 of the mdx mouse, which contains the point mutation, could be skipped in up to 70% of the muscle fibers. If it were as successful in children, this technique has the potential to ameliorate the dystrophy of Duchenne patients.

Upregulation of utrophin

Utrophin is similar to dystrophin and present also in Duchenne patients. Increasing its amount by activating its gene may delay muscle degeneration without immune rejection.

Upregulation of utrophin: In the laboratory of Kay Davies (Oxford), experiments were undertaken for many years to increase the amount of the protein utrophin at the muscle cell membrane, to upregulate it, because this protein is similar to dystrophin. It is present in many body tissues, also in muscle, but there it is concentrated near the neuromuscular junctions, the regions where the motor nerves contact the muscle membrane. Although it is present only in small amounts, utrophin makes the Duchenne symptoms in humans and animals less severe than they would be if it were also missing. In fact, mdx mice, whose utrophin gene was removed experimentally, and which, therefore, have neither dystrophin nor utrophin, are really sick in contrast to the normal mdx mice whose muscles do not degenerate like in Duchenne boys. In order to show that utrophin, if it is present in sufficient amounts, can take over the functions of dystrophin and practically cure the muscular dystrophy of mdx mice, transgenic mice were made by introducing utrophin minigenes or full-length genes into their germline, a technique that cannot be used in humans, or by transferring the utrophin gene at birth with adenoviruses. The results are encouraging since the presence of utrophin at birth prevented the onset of the disease symptoms, and even at later stages some effect was seen.These experiments showed also that too much utrophin in muscles or in other tissues is not toxic and that there is no immune rejection because it is normally present in small amounts and thus known to the immune system. To determine the requirements for a future upregulation of utrophin in Duchenne boys including its timing, other transgenic mice were created in which the activity of the utrophin gene can be regulated by adding the antibiotic tetracycline to their drinking water. To find other substances which could upregulate utrophin, a new test system was developed.

Upregulation of utrophin by steroids: Thomas Meier (Basel) discussed experiments with isolated human muscle cells to determine how steroid drugs influence the production of utrophin. In these in vitro studies, one of these drugs, prednisolone, increased the amount of utrophin by about 40% within 4 to 5 days. Further experiments showed that prednisolone does not influence the amount of utrophin by acting on the promoter of the utrophin gene, but probably either stabilizes the utrophin molecule or blocks the action of proteases, enzymes which destroy proteins. Furthermore, substances with a structure similar to prednisolone were also used in these experiments. Some of them could upregulate utrophin equally well or even better than the original drug, and they are already commercially available for the treatment of skin diseases.

Standardization of measurements: In the laboratory of Jean-Marie Gillis (Bruxelles), the extent of the dystrophic signs in mdx mice are studied so that the results of different therapeutic experiments, e.g. the upregulation of utrophin, can be accurately documented. Besides the usual tests for creatine kinase and for general muscle force, many additional methods were developed or standardized, e.g. to measure the strength of individual muscles, their electric properties, and the amount of calcium in isolated muscle fibers. The experimental results could then becalculated in such a way that the recovery of a lost property, like muscle strength, can be stated in percent. As mdx mice in spite of the absence of dystrophin in their muscles show only very few outward clinical signs of the disease, such standardized methods are important for the comparison of experimental results from different laboratories.

Other proteins

The mutation of the dystrophin gene and the absence of dystrophin changes the activities of many other genes.

Proteins associated with dystrophin: Derek Blake (Oxford) discussed the role of alpha-dystrobrevin for the development of Duchenne dystrophy. This protein is part of the dystrophin-glycoprotein complex and has a structure similar to dystrophin. When it is missing in mdx mice, the other proteins of the dystrophin complex are not affected as in the Duchenne type of dystrophy but a dystrophy develops anyway. This may mean that dystrobrevin has something to do with the transfer of molecular messages within the cells in which other still unknown proteins are involved. By using special methods to look for proteins which would bind to dystrobrevin, two novel proteins, syncoilin and dysbindin, were identified. Dysbindin was also found to bind to alpha-actinin, a component of the contractile apparatus of the muscle, and to a new not yet characterized protein. The exact role of alpha-dystrobrevin in the interaction of these different proteins has still to be established. With the present results, however, it does not seem to be likely that upregulating alpha-dystrobrevin above its normal level would contribute to a Duchenne therapy.

A dystrophic worm: Experiments with an unusual dystrophic animal, the worm Caenorhabditis elegans, were reported by Laurent Ségalat (Lyon). These about one millimeter long worms are used extensively by gene researchers because not only are all their 19,733 genes known but also all their 959 body cells, 95 of which are muscle cells. They have muscles with a dystrophin similar to that of humans which also can have mutations causing muscular dystrophy. In order to find out in what way the different components of the muscle cell influence the development and the maintenance of a healthy muscle fiber, the activity of many of their genes were destroyed, one at a time, and the effect observed. An important observation was that the inactivation of the worm’s dystrophin and of some associated proteins led to muscle degeneration, and this degeneration could be delayed by the upregulation of dystrobrevin, a very short version of dystrophin, and by some other parts of the dystrophin complex. Further research could lead to the identification of still unknown components of muscle which would be able to slow down the muscular dystrophy in children.

Ativities of thousands of other genes: Gert-Jan van Ommen (Leiden) and his colleagues are using the new technique of expression profiling to determine which other genes are affected when the dystrophin gene is mutated and the muscle cells cannot make any dystrophin. With this technique, the activities of the genes were analyzed in the muscles of normal and mdx mice, of healthy and dystrophic boys, and most importantly, of transgenic mice which had neither their own dystrophin nor any utrophin, but which were genetically changed so that they had the entire human dystrophin gene with its 2.4 million genetic letters in all of their muscles. The results showed that the missing dystrophin causes many genes in the muscles to increase or to decrease their activities. A number of genes responsible for the energy production were decreased in these mice without dystrophin and utrophin, which means that their muscles have an energy crisis which contributes to the degeneration of muscle tissue in Duchenne dystrophy. On the other hand, many genes involved in the development and regeneration of muscle fibers were more active, some of them more than 100 times above normal. Similar results on gene activities in human dystrophic muscle were published one year earlier by the team of Eric Hoffman (Washington) and presented at the workshop by Diana Escolar. In addition, the Dutch researchers found that in mice the activities of many more genes were changed, of genes responsible for building the cell surface structures, for making signaling factors for protein synthesis, for intensifying immune reactions, and for other processes responsible for the clinical manifestation of the disease. In Duchenne boys, these activity changes were much smaller. In the mdx mice with human dystrophin in their muscles, the gene activities were normal in contrast to the mdx mice without dystrophin. This result confirmed that the presence of the human dystrophin can compensate for the missing mouse dystrophin and thus cure its dystrophy. Research using this new and powerful technique is only at its beginning. Future results will let the scientists better understand the complex relationship between the many components of the muscle architecture and its changes when dystrophin is lost. This should open new ways to the development of a therapy for Duchenne dystrophy.

Clinical trials

In France, the first gene transfer trial with Duchenne patients has started. Two steroids were compared in Germany. An international collaboration studies a number of possibly therapeutic substances in Duchenne boys. In the United Kingdom, two boys receiving prednisolone were clinically studied for more than 6 years and a large steroid study is being prepared.

Gene transfer trial with Duchenne boys: Serge Braun (Strasbourg) and Norma Romero (Paris) reported on a first gene transfer experiment with Duchenne patients performed in collaboration of the company Transgène in Strasbourg, the Hôpital de la Salpitière in Paris and the French muscular dystrophy association AFM. After testing several gene transfer methods in dystrophic mice and dogs, it was decided to use the full-length dystrophin gene, i.e., its entire cDNA, together with a strong promoter from a virus, in a plasmid as vector. Plasmids are small rings of DNA often present in large numbers inside cells. They have the advantage of not containing any protein and thus should not cause an immune reaction. The therapeutic gene to be transported has no protein either, it is pure or naked DNA. In further preliminary experiments with muscle cell cultures and mice, it was shown that this vector construction caused the appearance of new dystrophin at its correct place underneath the muscle cell membrane, that it restored the dystrophin-glycoprotein complex, and that it prolonged the life of the cells, mainly by reducing the increased amount of calcium. After these preparations, the first clinical study was started with nine Duchenne patients older than 15 years so that they could give their informed consent. None of the patients will have any therapeutic benefit from these experiments. During the study, the vector is injected into one muscle of one forearm. With this study, the researchers wish to find out whether the procedure is safe, i.e., whether there will be an immune reaction or an inflammation, and whether new and normal dystrophin will appear at the correct places in the fibers of the muscle which had received the plasmid vector. The French researchers are also working with the team of Jon Wolff in Madison, WI, who injected plasmid constructions with genes of a marker protein, which is easy to detect, into the blood circulation of limbs of rats and monkeys under pressure and found that afterwards up to 40% of the muscle fibers contained the transferred marker protein. Both laboratories are now testing the delivery of the naked dystrophin gene with plasmids directly into the blood circulation of dystrophic dogs.

Comparison between prednisone and deflazacort:

Bernd Reitter (Mainz) discussed his experiences with a large-scale clinical study in Germany between 1992 and 1997 in which 14 clinical centers participated. The aim of the study was to determine whether the new steroid drug deflazacort had the same muscle preserving properties and possibly fewer side effects compared to the similar steroid prednisone. The daily amount of prednisone given was 0.75 mg per kg body weight or 0.9 mg of deflazacort. With both drugs, the muscle force could be maintained for at least two to three years and ambulation in isolated cases until the age of 14. Deflazacort did not increase the appetite as much as prednisone did, therefore, the weight gain was significantly less. However, deflazacort caused a few more cataracts, slight turbidity of the eye lenses. After the end of the study, the treatment with deflazacort or, in some cases, prednisone, of most of the participating and new boys is continued as an open study in five of the original clinical centers and documented centrally using the same criteria as during the original study. The clinical courses of more than 100 Duchenne patients are on record with treatment periods of between a few months and more than 7 years. Some of the boys could largely preserve their muscle force, however at the cost of slower or arrested growth and other less important side effects. The study showed also, that the treatment results can be different from case to case. The cause of this variation has still to be investigated as well as the optimal time to start the treatment, questions which the present open long-term study will help to answer. Patients who wish to receive deflazacort should do this only within a well controlled study like the German one. A treatment outside a strict structure of controls cannot yet be recommended.

Large scale steroid trial planned: In the United Kingdom, the steroid trial group consisting of 16 pediatric neuromuscular centers with Francesco Muntoni (London) and Adnan Manzur as leading scientists, is contemplating a large therapeutic double-blind placebo-controlled trial with a low dosage of prednisolone which would be given daily for 10 days followed by 10 days without medication. The purpose of the study will be to determine whether under these conditions prednisolone is able to prolong ambulation. Statistical calculations have shown that for such a trial at least 190 Duchenne boys would be necessary whose recruitment would take three years while the trial itself would last about eight years. To shorten the duration of the trial to about five years, the ability to rise unaided from the floor will be used to assess the effect of the drug instead of prolongation of walking which is difficult to measure objectively. As the maintenance or even the improvement of the quality of life of the patients should also be an aim of a medication, methods to document such effects will be developed and then used in this trial.

International clinical trials: Diana Escolar (Washington) summarized the clinical trials she is organizing with the Cooperative International Neuromuscular Research Group in cooperation with many clinical centers in the United States and other countries. These centers are performing clinical trials with Duchenne boys to determine whether substances which were found to be active in experiments with mdx mice, may also positively influence the course of the dystrophy in children. For a substance to be tested directly in Duchenne children based on results on mdx mice, it has to meet several conditions: It should be safe and not toxic, should have no intolerable side effects, there should be a scientific reason to use it against Duchenne dystrophy, and it should have been approved in the United States or in another country as a drug for other diseases in children. If one of these conditions is not met, the potential drug should not go directly from studies in mice to human trials, but first should be tested in the dystrophic dog to get a better understanding of how it would influence the human disease. And the new method of expression profiling should be applied before and after the application of a potential drug in order to determine the exact drug action on the different components of the dystrophic muscles. Information obtained in such experiments could help to modify promising substances, if necessary, to avoid negative effects. The substances that are currently being tested were found two years ago in experiments with mdx mice. Although these mice without dystrophin do not show dystrophic signs like Duchenne boys, their muscles can be made weaker by exercise, for instance by putting the mice into a treadmill and forcing them to run for a certain time. The decrease of the muscle force can then be reliably measured. With this method, a number of rather simple compounds like creatine, glutamine, oxatomide, and coenzyme Q10 were found which are now being tested in Duchenne boys. Trials with four other substances, taurine, carnitine, and nicotinic acid, are planned for 2002. For 2003, pentoxifylline will be studied and another trial is planned to compare the positive and negative effects of prednisone when it is given every day or only on two days each weekat a higher dosage. In all trials, standardized methods are used to measure objectively the clinical and molecular effects. Secure internet connections exist for the coordination of the activities of all participating centers and for the collection of data and other information as soon as they become available. This makes this international collaboration very efficient and less costly.

Clinical evalutation of a steroid treatment: Victor Dubowitz (London) showed that careful clinical observations of patients can help scientists to decide how to apply a potential therapeutic drug without the aid of modern medical methods. Two 4-year-old Duchenne boys were given low doses of prednisolone, not on every day, but either during the first 10 days of each month only or first for 10 days followed by 10 days without medication. The forces of up to 32 different muscles were regularly measured for five years and statistically evaluated. Both boys had a dramatic response in remission of their actual signs and became able to jump, to hop, to go upstairs, and to get up from the floor without a Gowers’ sign, i.e. without pushing themselves up on their knees with their hands. In one boy, this situation was sustained for five years before he started to decline and to lose the ability to walk within one year. The other boy continues almost completely to be fully active after six years, and he is now over 10 years old. As far as their height, weight, and their bone condition are concerned, both boys developed quite normally. That means, there might be an optimal early time to begin this treatment, and that an “on-off” treatment with a low dose of prednisolone is well tolerated. After more studies of this kind have been performend, this treatment can probably be recommended for long-term use in Duchenne children.

Pharmaceutical studies

How do steroids work and how does one look for more compounds which may have therapeutic properties?

Changes of gene activities after steroid treatment:

Francesco Muntoni’s group (London) obtained preliminary results, also with gene arrays, about the reason why steroid drugs, prednisone, prednisolone, and deflazacort, delay muscle degeneration. While part of their action likely involves their anti-inflammatory properties, other mechanisms of action are also possible. By analyzing more than one thousand genes in mdx mice before and after treatment with prednisolone, it was found that a large number of genes showed reduced or increased activities. Among them were genes responsible for the structure of the muscle fiber, for the delivery of molecular signals into and inside the cells, and for immune reactions. An unexpected result was that the gene activity pattern in untreated 7-week old mice was different from that of 12- week old mice. Further analysis of the gene activities induced by steroids will help to understand their mechanism of action which might help to develop a more specific treatment with fewer side effects.

Looking for more active substances: In the laboratory of Annamaria De Luca (Bari), more methods were developed to measure reliably the manifestation of the disease in mdx mice, e.g., changes of contraction caused by the increased amount of calcium inside the muscle cell and the damage to a protein structure, an ion channel, which allows the passage of electrically charged chlorine atoms across the cell membrane. These methods measure effects on a more basic level than testing of muscle force alone can do. By this approach, differences of the effects of the experimental substances were discovered. For instance, although creatine had a positive effect on muscle force, it did not normalize any of the cellular properties studied, whereas taurine was only partly effective on the chloride transport but fully effective on the calcium-dependent contractile function and muscle strength. Two other substances, prednisone and IGF-I, a growth factor important for muscle regeneration, were active in repairing both kinds of damage. These new and precise methods will enhance the possibility of finding more substances, which would act on muscle structures influenced by the disease, and which, therefore, should also be tested in clinical trials with Duchenne patients.

Green tea and other promising substances: The research team of Urs Rüegg (Lausanne) uses myotubes from mdx mice in cell culture to search for potential therapeutic substances. One of the effects of the missing dystrophin is the abnormal increase of the amount of calcium in the muscle cells. Experiments with the myotube system showed that creatine normalized the level of calcium. Other substances were also investigated like steroids, e.g. prednisolone. The protein aequorin, which is especially sensitive to calcium, is now used to monitor changes in the amount of calcium in the myotubes. Another approach was to add green tea extract to the food of mdx mice. Green tea is known to have antioxidant properties, i.e., it prevents damage caused by reactive oxigen which is known to contribute to muscle dystrophy. After the green tea treatment, the degeneration of some muscles was slower than that of the same muscles in untreated mdx mice, an effect that has also been observed with creatine.

The future

What happens when an effective drug is found, and when will there be a therapy?

When an effective drug is found, how will it reach the patients? At the end of the meeting, Karin Bakker of a consultancy firm in Holland discussed what should be done when finally an experimental result is so promising that it should be used for making a therapeutic drug or method available to all Duchenne patients. The scientists should first protect their discovery by patents according to the rules established by their university. They should then decide to either set up a new company, or to work with an established pharmaceutical company. In both cases, the financial and legal requirements should be considered taking into account that the commercialization of a therapeutic agent or method against the rather rare disease Duchenne muscular dystrophy could probably benefit from special tax and legal regulations, so-called orphan drug regulations. A positive long-term relationship with an industrial partner will depend on many factors, which should be openly discussed and made part of a formal agreement. In the interest of the many families with Duchenne boys, the scientists and the commercial partners should do their utmost to make such a collaboration work without delay.

When will there be a therapy? A final word from the author of this report: When the dystrophin gene was identified in 1986 and shortly afterwards the protein dystrophin characterized, the fast pace of genetic research gave rise to the hope that it would soon be possible to introduce a new gene or to repair the damaged one and thus cure the disease. This optimism was premature. The first studies in 1991 with the myoblast transfer technique showed that this method, which looked promising in mice, was ineffective in Duchenne boys. Now, more than 16 years after the detection of the gene, there is still no therapy for Duchenne dystrophy. As this report shows, research is being done in many laboratories with many different methods mostly applied in dystrophic mice, and some already in Duchenne boys. However, these studies are time consuming, and the approval of a treatment will take additional years. Although an impressive amount of research results have already been obtained, much more is needed before it will be possible to predict how long it will take until a safe and effective treatment is ready for Duchenne boys. The answer to this question is the most important one for the parents and their sick sons. It will probably take many more years, until Duchenne muscular dystrophy is defeated. This is less than what has been hoped for, that is the negative side of this difficult problem, the positive is that hundreds of capable and dedicated scientists are working on a cure: Therefore, it is certain that an effective treatment will be there, sooner or later.

Addendum

Explanation of some scientific terms.

Clinical studies have to be performed in several steps, phase I, the proof that the new treatment will not have unacceptable side effects. Phase II, to ascertain whether the treatment really improves or maintains muscle force, and phase III what the optimal dosis will be. Practically all these studies have to be performed as double-blind trials, i.e., only half of the patients receives the substance to be tested whereas the other half receives an inactive compound, a placebo, and neither the patients nor the researchers know which patient belongs to which group until the trial is completed and the results are analyzed. Most of the experiments are performed with the dystrophic mdx-mouse, which has a point mutation at nucleotide 3,185 in exon 23 of its dystrophin gene. This mutation has changed a CAA codon, which signifies the amino acid glutamin, to a TAA codon, which is a stop codon so that the mouse has no functional dystrophin in its muscles. Because their connective tissue does not proliferate, these have no fibrosis and do not lose their muscles. However, they have certain symptoms which can be exactly measured. Although therapeutic methods can be studied with them, any results obtained must be confirmed with additional clinical studies in Duchenne patients. A child is not a big mouse! Stem cells exist in many body tissues, also in skeletal muscles and in bone marrow. They are non-specialized cells that can develop into many kinds of specialized cells, e.g. bone marrow stem cells into different types of blood cells and muscle stem cells into new muscle cells. These pluripotent cells are somatic or adult stem cells in contrast to embryonic stem cells which are totipotent and thus can develop into all kinds of body and germ cells. Stem cell research to find a therapy for Duchenne dystrophy uses only adult stem cells of experimental animals, thus the ethical problems connected with the use of human embryonic stem cells will be avoided. In order to transfer a gene, a transporter, a gene vector is needed. One way to transfer genetic material into living cells is to pack it into viruses which consist of a string of their own genes enveloped in a protein shell. They attach themselves to a receptor protein on the surface of a living cell, introduce their genes into the cell and then use, enslave, the synthesizing apparatus of the cell to reproduce themselves. Mainly two kinds of viruses are used as gene vectors in Duchenne research, the adenovirus, which normally only causes the common cold, and the ten times smaller adeno-associated virus. These viruses can no longer multiply inside the target cell because the scientists have eliminated almost all of their own genes. The most advantageous virus seems to be the gutted, practically empty adenovirus, which does not contain any genes of its own, and thus has room for the 14,000 active genetic letters, the cDNA, with the information for the entire dystrophin protein.

A nucleotide, one of the bases with its sugar, ribose in RNA, or desoxyribose in DNA and phosphate, is a genetic letter. Several, less than about 100, connected together are called oligonuleotides. Gene profiling: To analyze the activities of thousands of genes in one single experiment, gene arrays are used. As the complete sequences of almost all the human genes are now known, thousands of short pieces of them, can be automatically made and robotically attached in a predetermined pattern on a quartz chip, a few centimeters on each side, where they cause points of light when they combine with synthetic DNA copies of messenger RNA from a sample of muscle tissue. As only those genes, which actively make their messenger RNA, lead to the production of light, one can automatically record this light and determine with computer programs which genes are turned on and to what extent and which are turned down. Transgenic mdx mice are a very powerful research tool because one or more genes can be added or removed, and by studying the effect, one can deduce the function of the added or removed gene. To add a gene, a gene is injected into individual cells of a very early embryo, the blastocyst stage. To destroy a gene, a knock-out mutation is caused with genetic methods at the same early development stage. The very few animals which are then born with the additional gene in one of their chromosomes or with one of their own genes knocked out can then be raised and multiplied. For obvious reasons, these genetic experiments cannot be done with humans.

In the years 1990 and 1991, a whole series of clinical trials with Duchenne boys were undertaken with the myoblast

transfer technique which had shown positive results in mdx-mice: myoblasts, muscle precursor cells, which can develop into mature muscle cells, from a healthy donor thus containing the normal dystrophin gene, were injected in many places directly into dystrophic muscles. These experiments were not successful in Duchenne boys, because the myoblasts did not migrate sufficiently from the injection sites, there were immunological problems and, above all, almost all of the injected myoblasts had died after a short time. Because the genetically inactive introns of a gene are much longer than the active excons, DNA without the introns, that is, with the exons joined, are needed for genetic experiments, especially for gene transfer studies. This so-called cDNA, complementary DNA, can be made by using the enzyme reverse transcriptase which uses the genetic information of the messenger RNA of a gene, containing only the transcribed introns, to make double stranded DNA. In the example below, it would mean making the DNA of lines A and B by using the RNA of line C as a template, as an original text to copy. Most of the proteins are enzymes, biocatalysts, which guide practically all processes of life like development, growth, digestion, muscle contractions, etc. They have a specific three-dimensional structure which arrange the partners of a biochemical reaction in such an optimal way that they react with each other at body temperature. Biological experiments are made in vivo, when living organisms are used, or in vitro, when isolated parts of organisms, like muscle fibers, are studied in the laboratory, e.g. in cell culture, in glass dishes or other containers.

Example of a point mutation in the dystrophin gene, leading to a premature stop codon.

The following example shows how the deletion of one base pair produces a premature stop codon in exon 2 of the dystrophin gene. The codons 1, 2, 3, and the G of codon 4 belong to the end of exon 1, the remainder to the beginning of exon 2.

Normal dystrophin gene and protein:

Codon and amino acid position:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Both strands of DNA, beginning of the coding region of the dystrophin gene, parts of exons 1 and 2, introns not shown:

A ATG-GAA-GAT-GAA-AGA-GAA-GAT-GTT-CAA-AAG-AAA-ACA-TTC-ACA-AAA-TGG-GTA-AAT-GCA-CAA-TTT

B TAC-CTT-CTA-CTT-TCT-CTT-CTA-CAA-GTT-TTC-TTT-TGT-AAG-TGT-TTT-ACC-CAT-TTA-CGT-GTT-AAA

Messenger RNA, reading frame not disturbed, in-frame:

C aug-gaa-gau-gaa-aga-gaa-gau-guu-caa-aag-aaa-aca-uuc-aca-aaa-ugg-gua-aau-gca-caa-uuu

The first 21 correct amino acids of dystrophin:

D Met-Glu-Asp-Glu-Arg-Glu-Asp-Val-Gln-Lys-Lys-Thr-Phe-Thr-Lys-Trp-Val-Asn-Ala-Gln-Phe

Dystrophin gene with point mutation, messenger RNA with premature stop codon:

Both strands of DNA, point mutation in codon 10, deletion of base pair AT: E ATG-GAA-GAT-GAA-AGA-GAA-GAT-GTT-CAA-AGA-AAA-CAT-TCA-CAA-AAT-GGG-TAA-ATG-CAC-AAT-TTT F TAC-CTT-CTA-CTT-TCT-CTT-CTA-CAA-GTT-TCT-TTT-GTA-AGT-GTT-TTA-CCC-ATT-TAC-GTG-TTA-AAA Messenger RNA with deletion of a at position 10, shifted reading frame, out-of-frame, premature stop codon at position 17: G aug-gaa-gau-gaa-aga-gaa-gau-guu-caa-aga-aaa-cau-uca-caa-aau-ggg-uaa-aug-cac-aau-uuu Protein, incorrect amino acids in positions 10, and 12 – 16, end of protein synthesis at position 17: H Met-Glu-Asp-Glu-Arg-Glu-Asp-Val-Gln-Arg-Lys-His-Ser-Gln-Asn-Gly-stop 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Abbreviations: Âases in DNA: A = adenine, C = cytosine, G = guanine, T = thymine. Bases in RNA: a = adenine, c = cytosine, g = guanine, u = uridine. Amino acids: Asp = aspartic acid, Asn = asparagine, Glu = glutamic acid, Gln = glutamine, Gly = glycine, His = histidine, Lys = Lysine, Met = methionine, Phe = phenylalanine, Ser = serine, Thr = threonine, Trp = tryptophan, Val = valine. Lines A to D: Normal dystrophin gene without a mutation. Line A is the DNA sequence of the normal gene starting with the ATG codon, which is the genetic signal to start the synthesis of a protein with the amino acid methionine. Line B is the other strand of the DNA with the complementary base sequence: A always faces T and C faces G. The hyphens are only there to indicate the reading frame, in reality, there are no spaces between the codon triplets. When the gene is active, when it is transcribed, the first product is the pre-messenger RNA, which contains also the usually very long introns between the exons. For reasons of space, this is not shown here, because the intron between exons 1 and 2 is 191,081 base pairs long. The combined parts of the exons 1 and 2 are shown in line C, the messenger RNA. As this is RNA and not DNA, the abbreviations of the bases are written in lower case letters. This sequence is complementary to the DNA bases in line B but with u, uridine, instead of thymine. Each of the three-letter codons contains the instruction to add one of the 20 different amino acids to the growing protein chain. According to the universal genetic code, the 21 codons give rise to the first 21 amino acids of the dystrophin protein, shown in line D with the abbreviations of their names. The process of reading the code words and assembling the amino acids in the ribosomes is called translation. Lines E to H: Gene with a point mutation. Lines E and F are the same as lines A and B with the exception of the deletion, the removal, of the A-T base pair in the middle of codon 10. This changes codon 10 in the messenger RNA from aag to aga and shifts the reading frame by one position, so that the first base of codon 11 is now the third base of codon 10. But aga does not direct lysine, Lys, to be added to the growing protein chain but arginine, Arg. And all the following code words are also shifted one position to the left by the out-of-frame mutation. This leads to different, incorrect, amino acids in postions 12 to 16, while the amino acid in position 11, lysine, remains the same because the corresponding code word, aaa, is not affected by the frame shift. However, instead of the codons -guaaau- at positions 17 and 18 of the normal messenger RNA, the codons are now -uaa-aug-. And uaa is not a codon for an amino acid but a stop codon which was hidden in the two normal codons 17 and 18, underlined in line C. It is a premature stop codon. At this point, the protein is truncated, its synthesis stops prematurely, the incomplete protein cannot fulfill the normal function of dystrophin and Duchenne muscular dystrophy develops.

Participants of the Workshop
The scientists are listed alphabetically with their abbreviated addresses and without titles.
Many of them are professors and all have an MD or PhD.
Karin Bakker, PharmaPlus Consultancy BV, The Netherlands
Derek Blake, Dept. of Pharmacology, University of Oxford, Oxford
Serge Braun, Transgène S.A., Strasbourg
Jeffrey Chamberlain, Dept. of Neurology, University of Washington, Seattle, WA
Judith van Deutekom, Dept. of Human Genetics, Leiden University Medical Center, Leiden
Kay Davies, Dept. of Genetics, University of Oxford, Oxford
George Dickson, Royal Holloway College, University of London, Egham, Surrey
Victor Dubowitz, Imperial College School of Medicine, Hammersmith Campus, London
Diana Escolar, Children’s National Medical Center, Washington, DC
Michel Fardeau, Institut de Myologie, Paris
Jean-Marie Gillis, Dept. de Physiologie, Université de Louvain, Bruxelles
Robert Kapsa, Melbourne Neuromuscular Research Institute, Melbourne
Hanns Lochmüller, Genzentrum der Universität, München
Annamaria de Luca, Dipt. Farmacobiologico, Università de Bari, Bari
Fulvio Mavilio, Dept. of Biomedical Sciences, University of Modena School of Medicine, Modena
Thomas Meier, MyoContract Pharmaceutical Research AG, Liestal (Basel)
Francesco Muntoni, Dept. of Paediatrics, Imperial College School of Medicine, London
Giovanni Nigro, Academia Gaetano Conte, Napoli
Gertjan B. van Ommen, Dept. of Human Genetics, Leiden University Medical Center, Leiden
Terrence Partridge, Muscle Cell Biology Group, Hammersmith Hospital, London
Thomas Rando, Dept. of Neurology, Stanford University School of Medicine, Palo Alto, CA
Bernd Reitter, Kinderklinik der Universität, Mainz
Norma Romero, Institut de Myologie, Paris
Urs Rüegg, Ecole de Pharmacie de l’Université, Lausanne
Laurent Ségalat, Université Lyon-1, Villeurbanne
Dominic Wells, Dept. of Neuromuscular Diseases, Imperial College of Medicine, London
Günter Scheuerbrandt, PhD
Im Talgrund 2, D-79874 Breitnau, Germany
Tel. +49-7652-91813-0, Fax +49-7652-91813-13
e-mail: gscheuerbrandt@t-online.de