Microsoft word - 2003-approaches.doc

Research Approaches towards a Cure
for Duchenne Muscular Dystrophy
A report on the state of international research for the development of
a causal therapy of Duchenne muscular dystrophy
Updated in August 2003
Written in cooperation with medical specialists and scientists
for the German Duchenne Parent Project
Aktion Benni & Co
and dedicated to all Duchenne boys and their families all over the world
by Guenter Scheuerbrandt, PhD
This report has been written for families who results are reported in a strongly abbreviated have one or more boys with Duchenne muscular dystrophy. It explains some basic scientific facts The first edition of this report in 2001 was and shows the now numerous approaches with based on an international workshop on Duchenne which research is trying to find a scientifically therapy research in May 2000 at the National In- justified and thus effective therapy of Duchenne stitutes of Health in Bethesda near Washington. muscular dystrophy. Because scientists in more This third edition of the report was written in
than one hundred laboratories in many countries July and August 2003 mostly with information
of the world are working to find a cure for this from the literature and by correspondence with disease, only the most important of their results are described here in greater detail. Some further Introduction
Some basic scientific facts are explained, what genes are, how they work,
why dystrophin is important, how mutations cause Duchenne muscular dystrophy,
and how the disease is inherited.

Genes and their function: Genes are functional
units of the genetic material in the chromosomes Each of the about 100 trillion (100 x 1012) of each cell. This material is desoxyribonucleic body cells in a human being contains in its nu- acid, DNA. Its structure looks like an intertwin- cleus 46 chromosomes with a total of more than ed ladder, the double helix. It was detected by 6 billion genetic letters, grouped in about 25,000 James Watson and Francis Crick in 1953, 50 to 35,000 genes. Almost all details of the se- years ago. The two backbones or strands of the quence of these letters are now known. It is the ladder are long chains of phosphoric acid and genetic information, which is passed on from desoxyribose, a kind of sugar. The rungs consist generation to generation with very little changes of four different chemical substances, the bases or mutations. These mutations, which were or genetic letters: adenine, guanine, thymine, and necessary for the evolution of all living beings, cytosine, abbreviated A, G, T, and C, two of can also have negative consequences as, e.g., which always face each other in one rung of the helix. For spatial reasons, the rungs can only Most of the genes carry the information for contain the pairs A-T or G-C. If the sequence of the construction of one or more proteins, which these bases on one strand is e.g. consist of amino acids. The sequence of the amino acids, of which there are 20 different the sequence on the opposite strand must be kinds, is important for the function of the pro- teins such as enzymes, the catalysts for bio- i.e., the sequences are complementary to each chemical reactions in the body, as regulators for length are produced. The main product is the In the cell nucleus, where the chromosomes full-length dystrophin, a very long protein con- reside, the genetic information of the genes is copied or transcribed to another genetic sub- Dystrophin is part of the costamers, which stance of a similar structure, the pre-messenger connect the Z discs of the sarcomers, the con- ribonucleic acid, pre-mRNA. The genes of mul- tractile structures, with the sarcolemm, the cell ticellular organisms consist of active sections, membrane. It is thus important for the mecha- exons, and inactive ones, introns. After the trans- nical stability of the muscle cells during muscle cription, the introns, which are often much lon- ger than the exons, are removed from the pre- Dystrophin network: Dystrophin belongs to
mRNA, and the transcribed exons spliced to- a network of many different proteins of which gether to the messenger RNA, mRNA, which is more than 50 are known. Among them are the then exported to the ribosomes, the protein syn- dystro- and sarcoglycans, the synthrophins and thesizing structures in the cytoplasma of the cell. integrins, dystrobrevin, nitric ocide synthase, and In the ribosomes, catalytic acting RNAs, ri- other components such as dysferlin, sarcospan, bozymes, use the genetic information of the laminin, caveolin, telethonin, myotolin, agrin, mRNA to construct specific proteins out of ami- neurexin, desmuslin, syncoilin, fukutin, aq- no acids which are delivered to the ribosomes by uaporin, spectrin, collagen, calpain and others. In another kind of RNA, the transfer RNAs or the future, more components are expected to be identified (Campbell, Iowa City). The RNAs use the base U, uracil, instead of When dystrophin is missing, the balance be- the very similar base T of the DNA. In the tween the different parts of this dystrophin com- mRNA, three consecutive genetic letters always plex is disturbed. Especially the dystroglycans, signify one of the 20 different amino acids ac- the sarcoglycans and sarcospan are reduced or cording to a genetic dictionary, the genetic code, disappear completely. Every one of the proteins which is the same for all life on earth. Thus, the of the complex has its own gene which also can genetic script uses only four letters, and its be disturbed by mutations. This leads to the words, the codons, are always three letters long, presently known 13 different limbgirdle and 5 triplets. There are no spaces between the words, congenital muscular dystrophies, as well as to at and three different stop codons exist, UAA, UAG, and UGA, where the protein synthesis is How big are molecules, DNA and dystro-
phin? A lay person seldom has a correct idea of
Dystrophin gene: Duchenne muscular dys-
the sizes of molecular structures with which sci- trophy is one of the most frequent hereditary dis- eases. About one in 3,500 boys is born with this disease, which is caused by a mutation or dam- strates the smallness of a simple molecule. Pour age of the dystrophin gene with the consequence on quarter of a liter of wine at the straits of Gib- that the protein dystrophin is no longer present or raltar into the Mediterranean. Then mix the Me- exists only in traces in their muscle cells. diterranean well and, at the other end in Alex- The dystrophin gene was identified in 1986 andria, take out one quarter liter of water with on the X chromosome (Kunkel, Boston) and its the same wine glass. How many alcohol mole- structure elucidated shortly afterwards (Hoffman, cules will you find in the wine glass? Twenty Washington).With 2.6 million base pairs, it is the longest gene of man. Only 0,5 % of the base The DNA double helix has a diameter of two pairs, 13,973, belong to the 79 exons of the gene, nanometers, millionths of a millimeter. If one en- which contain the active coding sequence, the in- larges the helix to a diameter of one centimeter, formation for the synthesis of the different forms how tall would a person 1,80 meters tall be if of the protein dystrophin. The transcription of one would enlarge him/her by the same factor? the genetic information of the dystrophin gene Nine thousand kilometers, this is about the dis- into mRNA is under the control of five pro- moters, DNA regions governing the splicing pro- Each cell nucleus of the about 100 trillion cess so that a number of dystrophins of different (100 x 1012) cells of an adult human being con- tains the complete genetic material with 6 billion somes in the nucleus of every one of their body base pairs. The entire DNA in all chromosomes cells, one Y chromosome and one single X chro- of each single cell nucleus is two meters long! mosome. If the dystrophin gene on their X chro- Every dystrophin protein is 125 nanometers mosome is damaged by a mutation, it cannot be long, thus 80,000 of then laid down in a straight compensated for by an intact gene on a second X chromosome, as it is possible for mutations on There are 114 billion dystrophin molecules the autosomes, which are always present in pairs. and the same number of dystrophin protein com- Therefore, Duchenne and Becker dystrophy af- Mutation and origin of the disease: Du-
chenne muscular dystrophy is caused by three in their body cells. When they carry a mutated different kinds of mutations of the dystrophin dystrophin gene on one of their X chromosomes, gene: deletions, if one or more of the exons of they can transmit the disease, they are genetic the gene are missing, duplications, if parts are carriers. As one of the X chromosomes is inacti- duplicated, and point mutations, if single bases vated in a random fashion, about only half of are exchanged, eliminated or added. As the read- their muscle cell nuclei have an intact dystrophin ing mechanism of the information in the ribo- gene. This is sufficient to cause either none or somes always reads code words of three letters only weak clinical symptoms of the disease. one after the other without spacings, a mutation About two thirds of Duchenne boys inherit does not upset the reading frame if the number the disease because their mother is a genetic car- of letters missing or added can be divided by rier. At the meiosis, the cell division leading to three without a remainder. In this case, the dys- the egg cells, each egg cell receives only one X trophin made is longer or shorter. If this change chromosome. The probability that it is the X only involves non-essential structures of the dys- chromosome with the gene mutation is 50 %. trophin as e.g. the central part, it can still be part- Therefore on average 50 % of her sons will have ly functional. Then, the benign form of dystro- Duchenne muscular dystrophy and on average phy, Becker muscular dystrophy, develops. 50 % of her daughters will also be carriers. If the mutation shifts the reading frame by These risks remain the same for all children in a one or two bases, then a whole string of incorrect family, it is not smaller if the family already has amino acids is incorporated into the protein start- ing at the mutation site until finally a new and If the mother is a carrier, the mutation arose premature stop codon is reached which termi- either in the germ cells of her parents or in an nates the synthesis. The incomplete dystrophin earlier generation. As all body cells of a genetic cannot fulfil its normal function, it is degraded carrier have the mutated gene, her carriership can and Duchenne muscular dystrophy develops. be detected by a gene analysis in the leukocytes, Without dystrophin, the muscle cells degene- the white blood cells, which contain chromo- rate. They are continuously regenerated, but the repair mechanism eventually fails. The destroyed About one fourth of Duchenne boys have a muscle fibers are replaced by fat and connective new mutation. In these cases, the mutation took tissue leading to fibrosis and, at the age of two to place spontaneously in the particular egg cell of three years, to the first visible symptoms of the the mother that then became the patient. As only one egg cell is affected, all other children of Where the motor nerves contact the muscle these women do not face a greater risk than the membrane, another protein with a structure simi- lar to dystrophin, utrophin, is located and con- About one tenth of Duchenne boys have a tributes to some extent to the stability of the mother with a germ cell mosaic, because the new muscle membrane (Davies, Oxford). Without mutation arose early in the germ cell formation utrophin, the disease would progress much and the mutated cell developed into a group of egg cells each carrying the mutation. As more Genetics of Duchenne muscular dystrophy:
than one egg cell is affected, another son can in- In addition to the 44 normal chromosomes, the herit the disease too, or a daughter can be a gene- autosomes, boys have two different sex chromo- As present genetic methods cannot detect a of increasing contractures at the foot, knee, and germ cell mosaic, a prenatal diagnosis should be hip joints, the patients lose their walking ability offered during a second pregnancy to all women at 10 to 12 years. Increasing spine deformities, who already have a boy with Duchenne muscular scoliosis, and restrictions of movement make dystrophy, not only to those who are proven ge- them soon completely dependent on intensive netic carriers (Müller-Reible, Würzburg). care. The involvement of the respiratory and Clinical course of Duchenne muscular dys-
heart functions lead to death by cardiac and cir- trophy: The first clinical signs appear at about
culatory insufficiency at an early adult age. Early two to three years of age causing difficulties in orthopedic operations to avoid contractures and walking and especially in climbing stairs. With- spine deformities as well as respiratory aids and out early detection, even today the disease is ge- other measures can improve the quality of life nerally diagnosed at about 3 to 5 years. Because and significantly prolong life expectancy.
Gene diagnostics
Genetic methods for the diagnosis of Duchenne and Becker muscular dystrophy
in a boy, during a pregnancy at risk, and for the determination of carriership in a woman.
Early detection in infancy.

Diagnosis of Duchenne muscular dystrophy in
the 79 exons. A deletion is detected when one or a boy: For a gene analysis, a blood sample with
leukocytes is needed. These white blood cells, If no deletion is found, a point mutation is but not the red ones, have cell nuclei which con- probable. To characterize it unequivocally, the tain the hereditary material DNA. The white base sequences of most of the exons would have cells are isolated and the DNA is obtained from to be determined. Such sequence determinations, however, are very demanding, and they are not As about 60 % of Duchenne patients have yet routinely offered. In these cases, a final dia- deletions of one or more exons in the their dys- gnosis can only be made by investigation of trophin gene, one looks first for these deletions muscle tissue obtained by a biopsy. There, not which can be everywhere in the gene but which the dystrophin gene is analyzed, but the dystro- are more numerous in some regions than in oth- ers. Generally, in a first step, 19 selected exons In this method, after electrophoretic separa- of the 79 in the dystrophin gene are multiplied, tion, the protein pattern is transferred to another amplified. Normally, one does not amplify all carrier material by blotting, where it is made vi- these 19 exons simultaneously, but by multiplex sible with antibodies. Or fluorescent antibodies reactions in groups of 4 to 6 exons. are attached to the dystrophin, which can then be This already allows the detection of 98 % of detected under the microscope as a bright line all deletions. In two thirds of these cases, one around the healthy muscle cells. In Duchenne can already deduce whether the reading frame is dystrophy, it is not seen there or only in traces, disturbed or not and thus predict whether the and in Becker dystrophy, these lines are often patient has a Duchenne or a Becker dystrophy. frayed, interrupted and mostly much weaker than For the remaining third, more exons must be If a gene analysis gives an unequivocal result, The amplification is performed by the poly- a muscle biopsy is often no longer necessary. merase chain reaction, PCR, which needs prim- Especially, small children could thus avoid this ers for each exon. The primers are short synthe- surgical procedure. However, if a muscle biopsy tic DNA sequences which attach themselves to is necessary, then it can also be performed as a the beginning and the end of an exon sequence. needle biopsy under local anesthesia. The small gene fragments obtained in this way Prenatal diagnosis: For a prenatal diagnosis
are separated by electrophoresis where they mi- of Duchenne dystrophy, there generally must be grate different distances in a gel layer and then a definite reason mostly because there already is can be made visible as bands like unevenly spac- or was a patient in the family. For a diagnosis ed rungs of a ladder. Each band represents one of during early pregnancy, tissue of the unborn child must be obtained either by a chorion villi biopsy in the 10th to 12th pregnancy week or by If a boy has a deletion in the dystrophin gene, an amniocentesis in the 13th to 16th week. not only are one or more exons missing but also For a chorion villi biopsy, a small piece of the the markers in the introns between them. In a future placenta is aspirated through a fine canula. woman, one can check whether these markers If it is certain that cells of the child have been are also missing on one of her X chromosomes. obtained, the tissue can be used directly for the If the markers are missing which her son also analysis. The risk that this intervention termi- does not have, then it is highly probable that she nates the pregnancy involuntarily is about 3 to has the same deletion as he on one of her X chromosomes. She is thus a genetic carrier. If The advantage of an amniocentesis is the lo- she is not a carrier, she should have additional wer abortion risk of below 1 %. The disadvan- tage is that the few cells of the child in the amni- The DNA markers can also be used if the pa- otic fluid must first be multiplied in the labora- tient in the family does not have a deletion. Then tory, and this can last up to 3 weeks, therefore, one does not know what the mutation looks like. the analysis cannot begin before the 15th to 18th But based on the arrangement of the markers, one can say on which of the two X chromosomes If sufficient cells of the child have been ob- of the mother the mutated gene is located which tained, the chromosomes are investigated first. her sick son has inherited from her. And every This allows to determine the sex of the child. If it woman in the family who has the same X chro- is a girl, then, in most cases, no further tests are performed, because the consequences of a Du- This indirect method has been used for about chenne carriership should be discussed with the 20 years, one now knows, however, many more girl when she is old enough to understand them markers. Therefore, one can almost always dis- tinguish the two X chromosomes of a woman If the unborn child is a boy, then a gene ana- from each other, and it hardly ever happens any lysis if performed in most cases with the same more that a family is not informative. techniques as described for the diagnosis after If, however, all these methods are not suc- cessful, then, in some cases, one can mark cer- Diagnosis of carriership: If the mutation of a
tain gene regions on the X chromosome with flu- Duchenne boy in the family is precisely known, orescent gene probes and then check under the one can specifically look for the same mutation microscope whether light points are present or in the mother and in her female relatives. This is missing signifying normal or deleted gene regi- technically more difficult than in Duchenne boys ons. But this FISH method – fluorescence in situ because only one of the two X chromosomes of a hybridization – can only be used if certain spe- woman could carry the mutation. If she is a car- cific deletions have happened in the family. rier, the intensity of the bands after electrophore- Sequence analysis of the dystrophin gene
sis are only reduced to half of normal if one or as routine test: With the new analytic technique
more exons are deleted. However, the amplifica- SCAIP (single condition amplification/internal tion of the exons is not easy to control. And it is primer) it will in the future be possible to ana- often difficult to detect reliably the differences of lyze reliably within three days point mutations, small deletions, as well as all other mutations in Therefore, one often uses polymorphic mar- a routine fashion. In this method, all exons of the kers for the analysis. These are DNA sequences dystrophin gene, all intron-exon border regions in the introns between the exons which are al- with the splice signals, and all promoters are most always differently arranged on an individ- completely sequenced. All these gene regions, ual chromosome, and they also have different together about 110,000 base pairs long, are am- lengths so that the two chromosomes can be dis- plified with their special primers in one single tinguished. These marker sequences have noth- PCR reaction and then checked in a micro elec- ing to do with the disease, they are characteristic trophoresis. This allows the determination of all for each person, and they can also be amplified exon and promoter deletions, also those which by the PCR method and thus be identified un- are not found with the other methods. The base sequence of each gene fragment is then deter- 1974 until July 2003, more than 500,000 infant mined automatically with individual sequencing boys have been tested. Among them, 178 boys with strongly elevated CK activities were found, As this method also detects all mutations of for 136 of them, a Duchenne muscular dystrophy Duchenne carriers, it will lead to an improve- (1:3,700) was diagnosed or made probable, and ment of genetic counselling. Data files are now 28 had a Becker muscular dystrophy (1:17,800). compiled which will allow the prediction of the The early detection allows the parents to clinical course of a Duchenne or Becker muscu- make in time the decisions which are the correct lar dystrophy based on a defined deletion, dupli- ones for them, so that no other child with the cation or point mutation. For this purpose, many same disease is born in the same family or in ma- patients will have to be investigated clinically ternally related families. In addition, the diag- and genetically (Flanigan, Salt Lake City). nostic odyssey can be avoided which still often Early detection at infant age (without gene
takes many years until an expert for muscle dis- technique): In Germany, 4 weeks to 1 year old eases is found. Such programs will be necessary infant boys with Duchenne and Becker muscular in the future, because the progress in therapeutic dystrophy, which do not yet have clinical symp- research will lead to studies with very young pa- toms, are detected in a voluntary CK screening tients who have no clinical symptoms and whose program in which the enzyme creatine kinase, muscles are still largely intact (Scheuerbrandt, CK, is determined in a dry blood spot. From Therapeutic possibilities:
Research strategies for a causal therapy.
There is still no therapy for Duchenne muscular dystrophy.

To cure Duchenne muscular dystrophy, the con-
developed so far which would cure the disease of sequences of the mutation of the dystrophin Duchenne boys. Only three closely related drugs, gene, the muscle degeneration, have to be stop- prednisone, prednisolone, and deflazacort, have ped or at least alleviated. Research tries to do been found which, during a limited time, can this either by gene therapy or by drug therapy. slow down the degeneration of the muscles. Gene therapy means that either all exons of the Because of the severity of the disease, the pa- intact dystrophin gene, its cDNA, or parts of it, tients, their families, their doctors, and the public are introduced into each muscle cell, or that the in general are looking desperately for even the damaged gene is repaired by genetic techniques. smallest positive research result. And the media, A drug therapy would mean that a new or al- mainly newspapers and television, even the res- ready known drug is given to block or to slow ponsible among them, tend to exaggerate small down the muscle degeneration without influ- advances as important breakthroughs. But also encing the gene itself. On both approaches, im- the scientists themselves are often over-optimis- portant progress has been made during the last tic when they declare the results of their experi- ments with animals as therapeutic victories, thus There is no therapy yet: In spite of this pro-
raising false hopes. Not a single Duchenne boy gress, neither a gene nor a drug therapy has been Transfer of a new dystrophin gene
The exons of the entire or the shortened dystrophin gene can be transferred
into muscle cells by viruses, plasmids, or myoblasts. Immune reactions must be avoided.

It is reasonable to believe that the transfer, the
duce sufficiently large quantities of functional transportation, of sufficient quantities of the in- dystrophin which then migrates to its normal tact dystrophin gene into the nuclei of dystrophic place under the cell membrane where it is cor- muscle cells would cure the disease if the genetic rectly incorporated into the intricate dystrophin- information of the new genes is used by the pro- tein synthesizing ribosomes of the cell to pro- The dystrophic mouse: Most of the experi-
ments with this aim have been performed with formation for the complete dystrophin protein one kind of laboratory animal, the dystrophic and for additional control sequences. With these mdx mouse, which has a point mutation at base gutted viruses new dystrophin was produced in pair 3,185 in exon 23 of its dystrophin gene. This up to 30 % of the muscle fibers of a dystrophic mutation has changed a CAA codon, which sig- mouse followed by an improvement of muscle nifies the amino acid glutamine, to a TAA co- function in young as well as in older mice don, which is a stop sign, so that the synthesis of (Lochmüller, Munich; Chamberlain, Seattle). dystrophin is interrupted prematurely. Thus, the Two genes in one virus: In new experiments
mouse has no functional dystrophin in its mus- two mouse dystrophin cDNAs were packed into cles. However, these mice do not lose their mus- each gutted virus, that means, two times all 79 cles because they do not develop fibrosis, a pro- exons without the introns, and in addition two liferation of connective tissue, like Duchenne more gene sequences, the very strong enhancer boys do, so that the degeneration caused by the of the cytomegalovirus and the beta-actin promo- disease does not overtake the regeneration. ter. These gene vectors were injected into the Although gene transfer can be studied with tibialis anterior muscle of newborn mdx mice them, one should keep in mind that any results and into 4-6 weeks old juvenile mice. After 30 obtained with these mice cannot be regarded as days, 42 % of the cells in the treated muscle of immediately applicable to children as long as the newborn mice had new dystrophin which they are not confirmed by clinical studies with was still detectable after half a year. In the juve- Duchenne patients. A child is not a big mouse! nile mice, 24 % of the muscle cells had new dys- The dystrophic dog: Some experiments are
trophin after 30 days, which, however, was re- performed with dystrophic dogs, the golden re- duced to half the amount after 6 months. Only triever muscular dystrophy (GRMD) dogs the juvenile mice showed small immune prob- which, in contrast to the mice, have a muscular lems. At present, these new vector constructions dystrophy similar to the dystrophy of Duchenne are the most effective ones for the treatment of boys. They are really handicapped and hard to Duchenne muscular dystrophy by gene therapy raise and to manage. Their dystrophin gene has a point mutation in the splice receptor site of in- Gene transfer with adeno-associated vi-
tron 6 which leads to the deletion of exon 7 in ruses: These small viruses can only transport
the mRNA, to a frame shift, and a premature genetic material that is not longer than about 5,000 base pairs, about one third of the entire Gene transfer with adeno viruses: In order
dystrophin cDNA. Their advantage is that they to transfer a gene, a transporter, a gene vector, is transfer the gene more effectively than the nor- needed. One way to transfer genetic material into mal adenoviruses. The disadvantage is that the living cells is to pack it into viruses, which con- dystrophin cDNA to be transferred has to be sist of a string of their own genes enveloped in a shortened considerably to fit into this small vec- protein shell. They attach themselves to special tor. Patients with Becker muscular dystrophy, receptor proteins on the surface of a cell, inject that progresses much slower than Duchenne their genes through the cell membrane and then dystrophy, mostly have such shortened dystro- use the synthesizing apparatus of the infected phin in their muscles. Therefore, a transfer of one of these Becker mini genes would not com- Mainly two kinds of viruses are used as gene pletely cure Duchenne muscular dystrophy but vectors in Duchenne research, the adeno virus only transform it into the benign Becker form. and the ten times smaller adeno-associated virus. In order to determine which of the four re- The vectors derived from them cannot be further gions of the normal dystrophin protein – the two multiplied inside the target cell because almost end regions, the cystein-rich, or the central rod all of their own genes have been eliminated. The regions – are important and which are not, exten- most advantageous vector seems to be the gutted, sive experiments were performed with dystro- practically empty adenovirus, which does not phic mice which were genetically modified so contain any genes of its own, and thus has room that they had one of nine different shortened for up to 36,000 foreign genetic letters, sufficient for the entire cDNA, i.e., all exons with the in- It had already been known that some of the rodlike central region can be removed without function in a living animal, transgenic mdx mice loss of function. The detailed analysis of the were created which have an additional gene con- three-dimensional structure revealed now which struction inserted in their chromosomes. This particular parts of the rod structure could be de- consisted of an often used Becker gene which, leted without significantly losing the muscle pro- with 6,300 base pairs, is only half as long as the tecting function. One of the shortened dystro- normal gene, and which was preceded by a CK phins, which was about half as long as the nor- promoter of 1,354 base pairs. Shortened dystro- mal one, had practically the same properties as phin was then present during the entire life time the unaltered protein. Delivery of the genes of of the mice of up to 24 months in sufficient this and some of the other shortened dystrophins quantities to improve all measurable clinical with adeno associated viruses into the muscles of the mdx mice prevented and partially reversed The new protein appeared only in the muscles the dystrophic symptoms. These results demon- of the transgenic mice, significantly more in the strated that specific modifications of the dystro- fast muscles than in the slow muscles. The fast phin gene can generate novel proteins that are muscles, which can work immediately but not significantly smaller but more functional than the consistently, obtain their energy through glyco- naturally shortened dystrophins of patients with lysis, through the fast degradation of glucose Becker muscular dystrophy (Chamberlain, Seat- without the use of oxygen. The energy for the slow and consistently working muscles is pro- If about 30 % of the normal amount of un- vided by the enzymes of the respiratory chain, changed dystrophin re-appears, measurements on the slow “burning” of organic substances with the diaphragm of the mice indicated that the oxygen. As the fast muscles are destroyed first muscle function also improved. The transfer of by the muscular dystrophy, the predominant ap- the mini gene also led to an improvement of pearance of new dystrophin in these fast muscles muscle function. In addition, the gene transfer is able to ameliorate just this early consequence showed better results in younger animals, be- of the disease. Experiments have also shown that cause there were fewer problems of immune re- as few as 20 to 30 % of the normal quantity of jection, and, after a single injection, the newly dystrophin have a significant therapeutic effect. synthesized dystrophin remained in the muscles For these experiments with transgenic mice, longer than in adult animals. After a single treat- the Becker dystrophin gene together with the CK ment of newborn mice, newly formed dystrophin promoter was introduced into the germ cells by artificial insemination and genetic manipulation, For a future application in humans, this so that it was inherited to the progeny of the means that the patients would have to be treated mice. This is a technique which obviously cannot as soon as possible after birth when their muscles be performed with humans. To cure the sick chil- are still largely intact (Clemens, Pittsburgh). dren, one would have to transport the gene with Gene transfer via the blood stream: In the
its promoter by gene transfer with a vector soon experiments so far described, solutions of the vi- after birth, if possible, into all muscles (Loch- ruses that contain the dystrophin gene were in- jected directly into the muscles. In order to reach Amplification of targeted gene transfer:
all muscles, also those of the heart and the lungs, The viruses attach themselves to specific struc- experiments are being performed to develop a tures on the cell membrane, to the coxsackie and systemic treatment, i.e. the injection of the virus- adenovirus receptors, from where they penetrate es into the blood stream. To ensure that the new the muscle cell through the membrane. These re- dystrophin is only synthesized in muscle cells, ceptors, however, are more numerous on devel- another genetic sequence had to be added to the oping and regenerating muscle cells. They are dystrophin gene in the virus, the CK promoter, downregulated, less numerous, on mature muscle which normally activates the gene of the muscle cells which no longer divide. Therefore, gene protein creatine kinase. This promoter would transfer with adenovirus vectors is more efficient assure that the new dystrophin gene is activated To overcome this handicap, transgenic mice To investigate whether such strategies would were produced which expressed these receptors in large amounts on the surface of mature muscle transfer experiment can easily be monitored. fibers. This led to an up to 10 times more effi- When relatively large volumes of a solution cient transfer of the dystrophin gene (Holland, of these plasmids were injected under pressure into the arteries of the limbs of rats and rhesus As a multiplication of the of the virus recep- monkeys, the reporter genes beta-galactosidase tors on the muscle cells of a patient could only and luciferase were transferred into up to 20 % be achieved by additional gene technical meth- of the muscle fibers after one single injection and ods with all their risks, helper proteins were pro- into up to 40 % after repeated injections. The duced. In this case, they were monoclonal anti- pressure was created by blocking the venous out- bodies which consisted of only one kind of flow from the limb for a short time. Experiments molecule that could bind with one end highly to transfer the dystrophin gene are currently be- specifically on other, more numerous receptors. With their other end, these immune proteins GRMD dogs (Wolff, Madison; Braun, Stras- could bind to modified coat proteins of the adeno bourg). In France, clinical studies with Duchenne viruses. To achieve this binding, additional ge- boys have been started which are described in netic information for a chain of 33 amino acids from a bacterium was introduced into the adeno Experiments to overcome immunological
problems: As plasmids contain only DNA but
These adeno viruses could now attach them- not any proteins, gene transfer with such naked selves via the antibody bridge to integrin and DNA allows the examination of potential prob- nerve receptors (NCAM) which were much more lems with immune responses to newly created numerous than the adeno receptors. This method dystrophin without the interference of other pro- allowed up to 77 times more adeno viruses to teins which are newly produced with the other pass through the membrane into the muscle cell methods of gene transfer using viral and cell than before. With this new technique, which can also be used against other diseases, only the gene If the human dystrophin gene is introduced was transported which produces the easily de- into the muscles of mdx mice, an immune re- tectable protein beta-galactosidase. Experiments sponse develops against the foreign dystrophin. to transfer the dystrophin gene are being prepar- This does not happen after the transfer of the dystrophin gene of mice, although normal dys- Transfer of naked genes: For this technique,
trophin is not present in the muscles of these the genetic material DNA to be transferred is not dystrophic mice. The lack of an immune re- built into viruses but into plasmids, small circu- sponse to mouse dystrophin may be due to the lar DNA structures without protein, that exist in presence of the other forms of dystrophin. These bacteria where they mostly give rise to resistance results suggest that immune responses to dystro- against antibiotics. The advantage of this kind of phin gene transfer may not be a particular prob- gene transfer is that the plasmids do not contain lem in Duchenne patients, especially in those any protein, only genetic material, naked DNA, with point mutations that do not interfere with so that no immune reaction develops against the the synthesis of the other forms of dystrophin. Current experimental work is focussed on deter- Experiments were made with plasmids con- mining which of the other dystrophins must be taining marker or reporter genes for proteins that present to allow immune tolerance to the appear- can be detected in the muscle tissue after staining ance of new muscle type dystrophin after gene or by light production so that the success of a Experiments with stem cells
Among the satellite cells and the myoblasts of the muscles,
and also among the cells of the skin and the blood vessels,
are stem cells which can form new muscles or regenerate them.
Stem cells exist in many body tissues, e.g., also are non-specialized cells that can develop into in skeletal muscles and in bone marrow. They some kinds of specialized cells, e.g. bone mar- row stem cells into different types of blood cells new dystrophin was still present. In this case, and muscle stem cells into new muscle cells. there was no immune rejection although the re- These pluripotent cells are somatic or adult stem ceptor mice had not received any immune sup- cells in contrast to embryonic stem cells which are totipotent and thus can develop into all kinds As the muscle stem cells used in these experi- of body and germ cells. Stem cell research to ments were isolated from newborn normal mice, find a therapy for Duchenne dystrophy uses with this technique will probably not be applicable one exception only adult stem cells of experi- without modification with Duchenne children. mental animals. Thus the ethical problems con- They could, however, influence the recently re- nected with the use of human embryonic stem sumed myoblast studies (Huard, Pittsburgh; Stem cells from skeletal muscles: On the
Stem cells from bone marrow, muscles and
surface of the muscle cells are satellite cells, also skin: At the end of the last decade, the first ex-
called myogenic cells or myoblasts which, after periments with stem cells from bone marrow and an activation through several intermediate steps, with muscle satellite cells were performed. In all can form new muscles or repair injured muscles. experiments, the stem cells were obtained from To determine whether such “back-up” cells are normal male mice with normal dystrophin genes entirely or partly stem cells, satellite cells from and then injected into the tail vein of female mdx the skeletal muscles of newborn mice were iso- mice. The female mice were homozygous, that lated. It was found that they consist of three dif- is, the dystrophin genes on both of their X chro- ferent kinds, mainly the so-called EP and LP mosomes were mutated so that they could not myogenic cells, which are already quite specia- produce any own dystrophin. The fate of the in- lized. The third kind are muscle derived stem jected cells in the female mice could be followed cells, MDSC. They are very rare, only one by the detection of the male Y chromosome. The among 100,000 satellite cells is one such MDS female mice had to be lethally irradiated with X cell. But these rare cells are pluripotent, that is, rays to avoid an immune reaction. The transfer they can develop into muscle cells as well as into of the stem cells regenerated the bone marrow of cells of the nerves and of the blood vessels for new muscles. And they can be cultured for a First, 10 to 50 million cells from the untreated bone marrow were injected. Three months later, These MDS cells were multiplied in the labo- dystrophin could be detected in up to 10 % of the ratory, and then 400,000 of them in 25 microli- muscle cells of the mice. Some of the dystro- ters (a fortieth of a cubic centimeter) of liquid phin-positive cells contained Y chromosomes as injected in one single portion into one muscle of proof that the bone marrow cells from the normal living mdx mice. In order to see in what way male mice came via the blood stream and had these cells are different from the normal myo- fused with the muscle cells of the mdx mice. genic cells, the more specialized EP cells were They had brought with them the information for also injected into mdx mice under the same con- the normal dystrophin which was then used for the production of functional dystrophin. It was In the mice which were treated with the EP important to prove that the new dystrophin did cells, 130 muscle cells at the injection site con- not appear by reversion, that is, by spontaneous tained new dystrophin after 30 days, which how- exon skipping which occurs to a small extent in ever, after 90 days, had largely disappeared. In these mice, immune rejection by lymphocites To find out which part of the bone marrow was observed which was probably responsible cells has the stem cell properties, a small side for the slow disappearance of the new dystro- population, SP, could be separated from the ori- ginal cell mixture by the FACS method, fluores- In contrast to these results, 1,500 muscle cells cence-activated cell sorting. As these SP cells at the injection site of the mice treated with MDS had stem cell properties, they were used for fur- cells contained new dystrophin, about 10 times as many as after the injection of EP cells. Even Three months after the injection of only 2,000 after 90 days, in practically all of these cells, the to 5,000 of these SP bone marrow cells, up to 4 % of the muscle cells of the mdx mice had Mesoangioblasts, stem cells from blood
functional dystrophin. In a similar way, SP mus- vessels: One half million of newly detected stem
cle stem cells were isolated from muscle satellite cells from blood vessels of fetal normal mice cells, and 7,000 to 20,000 of them were injected were delivered by one single injection into the into the blood stream of female mdx mice. After artery of one hind leg of mice. These mice were one month, 5 to 9 % of their muscle cells con- missing one protein of the dystrophin complex, tained normal dystrophin and some also had Y the alpha sarcoglycan which causes one of the many limb-girdle muscular dystrophies in hu- Now, in 2002/2003, these experiments were mans. The injected cells migrated into the blood repeated and extended to stem cells which were capillaries, passed through their cell walls, and obtained from skin. The advantages of an isola- from there into all muscles of the injected leg, tion from skin are that this tissue is more easily accessible than muscle tissue, and that the accep- For at least three months after the injection, tor animals no longer have to be irradiated. new sarcoglycan was again present in almost A small side population of cells was also ob- normal quantities. The same was true for many tained with the FACS technique from skin cells of the other proteins of the dystrophin complexes which had the same surface properties, markers, which had also been missing. Also, there were as the SP muscle stem cells. The percentages of no immune reactions. After three consecutive SP cells were now 0.1 % from bone marrow injections every 40 days, not only was the gene cells, 0.7 % from muscle cells, and 1.2 % from defect almost completely corrected, but also the skin cells. Three months after the injection of muscle force in the treated leg was practically 6.000 to 50.000 of the SP skin cells, 2.3 % of the normalized again. Here as well, no immune muscle cells of the female mice contained new In order to check whether this technique This experiment had proved that stem cells could possibly be used for a gene therapy, the can be isolated from skin, and that, after appli- mesoangioblasts from “sick” mice were isolated, cation via the blood stream, they can form new and then the missing gene for alpha sarcoglycan muscle cells with normal dystrophin. This tech- was transferred with retroviruses into these de- nique is not yet sufficiently effective to be of fective stem cells. The systemic application of therapeutic significance. But after optimization, these ex-vivo treated cells led to the same posi- a Duchenne therapy might possibly be based on tive results as found with the normal cells. How- ever, retroviruses which insert themselves with Activation of muscle stem cells: Stem cells
the transported therapeutic gene into the chromo- with specific surface structures which are located somes, should not be used in humans because of in intact muscles, did not develop into new mus- cle cells, they were not myogenic. But if the For these experiments, the blood-vessel deriv- muscles were injured and the muscle cells regen- ed stem cells were isolated from unborn mice. erated themselves, then the number of muscle But if it were also possible to isolate them from stem cells increased up to ten times. These cells boys with Duchenne muscular dystrophy, these were now myogenic, they developed into unexpected positive results would mean that a myoblasts and new muscle cells, as experiments number of problems connected with the present with cell cultures and mice have shown. This ac- gene transfer experiments could be avoided as, tivation was initiated by Wnt proteins, a family e.g., low effectivity, immune rejection, and the of signal proteins which apparently are produced requirement of many injections into all muscles by injured muscle cells and which also play a role during embryonic development (Rudnicki, Intact dystrophin genes would have to be transferred into these patient-derived cells by an Some Wnt proteins have been isolated and ex-vivo procedure with the known vectors, then characterized now. They are about 400 amino multiplied in the laboratory, and finally re-inject- acids long and contain palmitic acid, a fatty acid ed into the most important arteries of the child. which seems to be important for the transmit- Possibly, the treatment would have to be repeat- tance of molecular signals (Nusse, Stanford). ed after several months, therefore it is important that these cells are not rejected by the immune system. And one of the most important advan- New experiments to transplant myoblasts:
tages would be that, with this technique, all mus- But work on this technique continues in order to cles could be reached, also the cardiac and respi- determine the reason why far less than 1 % of the ratory muscles. It would be a systemic treatment transplanted myoblasts survived in the dystro- with relatively few injections compared to other phic muscles. Researchers are now trying to gene therapy approaches (Cossu, Milano). characterize these rare active cells and to find out A stem cell experiment of nature: At the
how they can be isolated from the inactive cells. age of one year, a Duchenne boy had received a They are therefore looking for molecular signals, bone marrow transplantation from his father be- special substances in the muscle cells, which cause of another disease. As he could still walk could activate the myoblasts (Partridge, at 14 years in spite of the reading-frame-shifting deletion of exon 45, it was assumed that stem In earlier experiments, only two known drugs, cells from the bone marrow had provided the in- cyclosporin A and cyclophosphamide were used formation for new dystrophin. Investigation of to suppress the immune reactions in Duchenne new bioptic material, however, have now shown patients. Now, other substances have been that the milder symptoms of the disease are due investigated in studies with monkeys. This has only to a small extent to the transplant, but shown that the immune inhibitor FK506 alone or mainly to a spontaneous additional deletion of in combination with the inhibitor MMF avoids exon 44, so that, in the mRNA, exon 46 follows the rejection problems much better for several directly after exon 43. This exon skipping nor- months. A greater application density with injec- malizes the reading frame. Dystrophin can again tion distances of only 1 millimeter and a higher be produced but is shorter and therefore causes number of transplanted cells contributed to the the symptoms of a Becker dystrophy (see para- fact that in monkeys, up to 67 % of the muscle graph on exon skipping). Apparently, after 13 cells had taken up the myoblasts, they became years, a bone marrow transplantation can con- hybrid muscle cells (Tremblay, Québec City). tribute to some extent to a Duchenne therapy, A clinical study with the modified technique however not sufficiently so to significantly has been started in Canada (see section “Clinical change the disease (Kunkel, Boston). trials). Also in other laboratories, work is being Cell therapy with myoblasts: Muscles have
done to improve this cell therapy technique. E.g., their own stem cells, the satellite cells or myo- it was found that m144, a protein of the immune blasts, which, during the development or repair system, avoids the immediate death of the myo- of muscles, fuse together to form myotubes and blasts after transplantation into mouse muscles then long muscle fibers. As the word “myoblast transfer” has been misused, one now often pre- Experiments for an ex-vivo gene therapy:
fers to say myogenic cells instead of myoblasts. To avoid immune problems completely, experi- In the years 1990 and 1991, extensive clinical ments were performed to isolate the myoblasts studies with Duchenne boys were performed from the patient himself and then, in cell culture, with myoblasts. This myoblast transfer technique to transfer an intact dystrophin gene into the had shown positive results in mdx-mice. The cells, before they are re-injected again. In preli- cells used contained the normal dystrophin gene minary experiments an electroperforation tech- because they were derived from a healthy donor, nique was used to transfer the gene for a fluores- mostly from the father of the patient. They were cent marker protein into myoblasts in cell cul- applied in multiple injections at 0.5 centimeters ture. This technique transiently permeabilized distance into some muscles of Duchenne boys the membranes with a single electrical pulse, at with the expectation that muscle cells with nor- e.g. 400 volts across a distance of 4 mm. Under mal dystrophin would form. However, these ex- optimal conditions, the marker gene could be periments were not successful, because the trans- transferred into up to 70 % of the myoblasts planted cells did not migrate sufficiently inside where it produced the fluorescent protein. The the muscle, because there were immunological cells maintained their ability to fuse into myo- problems, and because almost all of the injected tubes, the next stage of muscle development myogenic cells had died after a short time Changing the genetic information (gene repair)
Mutations of the dystrophin gene on the X chromosome are repaired with oligoribonucleotides.
Entire exons are skipped in order to restore the reading frame.
Premature stop codons can possibly be skipped.

Experiments are performed with the aim not to
the protein region encoded by the missing exon introduce a functional dystrophin gene sequence within the full-length dystrophin protein which into muscle cells, but to change the faulty gene- was found localized correctly to the muscle tic information, thus repairing the mutation. membrane, and (3), most importantly, demon- Such a technique would have four important ad- stration that the gene on the X chromosome was vantages: (1) The risks of a virus-mediated gene corrected. The repair of the mutation was sus- transfer would be avoided, (2) not only the dys- tained for almost a year in this dog (Bartlett, Be- trophin of the skeletal muscles, but all other forms of dystrophin would also be repaired, (3) Gene repair in the mdx mouse: In a similar
the tissue-specific regulation of the dystrophin experiment, the point mutation in exon 23 of production would be maintained, and (4), manu- myoblasts from mdx mice was repaired in-vitro, facturing the therapeutic agents, oligonucleo- and these myoblasts then fused to myotubes tides, would probably be much easier and chea- which produced normal full-length dystrophin. per than making virus or plasmid vectors with Two weeks after one single injection of the oli- gonucleotides into the muscles of mdx mice, up Oligonucleotides are short specific DNA or to 2 % of the fibers around the injection site RNA sequences, which consist of a few bases contained new dystrophin which was not rever- connected to each other by ribose- or desoxy- tant dystrophin, i.e. normal dystrophin after a ribose-phosphate bridges of the nucleic acid spontaneous suppression of the mutation. This backbones. They can be manufactured auto- amount of new dystrophin remained stable for at least 10 weeks (Rando, Palo Alto). Three kinds of repair strategies are already Exon skipping: With this technique one tries
being applied: (1) repairing the mutation on the to change a Duchenne mutation into a Becker level of the gene itself, (2) changing the genetic mutation. This can be done by inducing the information during the splicing process of the splicing mechanism, which cuts out the introns pre-mRNA by exon skipping, and (3) ignoring a from the pre-messenger RNA, to also eliminate one specific exon after a point mutation or a de- Repairing the gene in the GRMD dog: At-
letion had shifted the reading frame and thus tempts to repair point mutations at the gene level caused a premature stop codon. The aim of this are made by using short chimeric oligonucleo- approach is to restore the disturbed reading tides, which contain RNA on one strand and DNA on the other. The DNA strand of the chi- The gene with its mutation is not altered by meric oligonucleotide is perfectly complemen- exon skipping, but the messenger RNA, mRNA, tary to the correct gene sequence at the point no longer contains the information of the skipped mutation site of the gene, while the RNA portion exon. As the mRNA is shorter than normal, the is perfectly complementary to the mutant se- dystrophin protein is also shorter, it contains quence. This leads to paired quadruplex struc- fewer amino acids. If the missing amino acids tures, fourfold strands, which are capable of cor- are part of the central region of the dystrophin, recting such a small mutation by activating the they are often not essential, and the resulting biological DNA repair mechanisms of the cell. shorter protein can still perform its stabilizing With this technique, a DNA and RNA oligo- role of the muscle cell membrane. The result nucleotide complementary to the splice-site mu- would be the change of the severe Duchenne tation in the dystrophin gene of the dystrophic symptoms into the much milder symptoms of GRMD dog was injected into a 6-week old af- fected dog. The treated muscle showed: (1) evi- Elimination of the mutation of the mdx
dence of restoration of the exon which was miss- mouse: This strategy was used to by-pass the
ing because of the mutation, (2) restoration of single base-pair defect, the point mutation in exon 23 of the mdx mouse. An antisense oligo- ess, the joining of the exons in the mRNA. ribonucleotide, consisting of 20 base pairs which Then, similar in-vitro experiments were per- were complementary to the RNA sequence of the formed with several antisense oligonucleotides pre-mRNA at the border region of exon 23 to in- specific to the analogous splice sites of the hu- tron 23, induced the splicing process to disregard man exon 46. With one of them, consisting of 19 the exon containing the mutation. The genetic in- base pairs, it was possible to delete exon 46 from formation of exon 23, which codes for 71 amino about 15 % of the dystrophin pre-mRNA in acids in the rod domain, was thus skipped during myotubes obtained from two Duchenne patients who had a deletion of exon 45. (The last page of This and the other investigated antisense-oli- this report shows the molecular details of this goribonucleotides were chemically modified, e.g., by protecting the normally free and sensi- This percentage of shortened mRNA without tive OH-groups of the ribose units of the RNA exons 45 and 46 led to normal quantities of a by methyl groups (-CH3). About 5 micrograms shortened dystrophin in at least 75 % of the myo- (millionths of a gram) of these stabilized poten- tubes. After 16 hours, this new dystrophin could tial “gene drugs” were injected together with the be detected in the cells, after 48 hours it had detergent F127 into the leg muscles of living moved to the cell membrane and stayed there for mdx mice. After two to four weeks, up to 20 % at least one week. The re-appearance of the dys- of the muscle fibers contained almost the normal trophin also led to the restoration of the dystro- amount of slightly shortened dystrophin. And, phin complex in and under the muscle cell mem- together with the other components of the dys- brane. In the meantime, the reading frame could trophin complex, it was located correctly on the be corrected in in-vitro muscle preparations from cell membrane. The muscle force was signifi- six other patients who had different deletions and cantly improved but not completely normalized. A repeated treatment increased the number of This technique is very specific, resulting only dystrophin-positive muscle fibers without the in the removal of the one targeted exon. With the development of an immune rejection (Wilton, same technique, it was possible to skip18 other exons in muscle cell cultures. Thus, this very Exon skipping in the mRNA of the human
promising strategy, proven in test-tube experi- dystrophin: Exon 45 of the dystrophin gene is
ments, may possibly later convert the Duchenne the single most frequently deleted exon in boys mutations into Becker mutations of more than with Duchenne dystrophy. This causes a frame shift in the mRNA and a premature stop codon Ongoing studies investigate different methods leading to a truncated and non-functional dystro- for the transfer of the of the antisense oligoribo- phin protein which is subsequently degraded in nucleotides into a living organism. To this end, the muscle cells. However, if both exons 45 and experiments with living mice are performed 46 are missing simultaneously, the reading frame which, instead of their own dystrophin gene, is not disturbed, not shifted, resulting in a shorter have the human gene in their muscles which, in than normal dystrophin with 108 non-essential addition, has been changed by creating “human” amino acids from the middle part of the protein deletions. Clinical trials on Duchenne boys with missing. Patients with this specific deletion have these methods can only be contemplated after the milder symptoms of Becker muscular dystro- these studies with “humanized” mice have given In-vitro experiments to specifically delete As the structure of the dystrophin gene is exon 46 from the dystrophin pre-mRNA in myo- known in all details, it is already possible to pre- tubes from mdx mice were successfully perform- dict which exon would have to be skipped in or- ed with four different antisense oligoribonucleo- der to restore the reading frame after a defined tides complementary to a splicing regulatory se- deletion or point mutation. At this time, such quence within exon 46. With these oligonucleo- predictions can only be purely theoretical. It is tides, an exon recognition sequence (ERS) or an not certain, whether the results obtained in cell exonic splice enhancer (ESE) is blocked, struc- culture or with mice, will be the same in Du- tures which are necessary for the splicing proc- chenne boys, and it is not certain either, whether, in an individual case, the restored but shortened introns during the splicing of the pre-mRNA dystrophin will really lead to the symptoms of a (exon skipping). As the point mutation of the Becker muscular dystrophy (van Ommen, van mdx mice is localized in exon 23, the removal of this exon had the consequence that the mdx mus- Splice sites are specific RNA sequences at the cle cells, which could not make any normal dys- borders of exons and introns which are essential trophin, now produced a slightly shortened dys- for the correct removal of the non-coding intron trophin protein which was localized at its correct sequences from the pre-mRNA. This pre-mes- senger RNA is the first product of an active gene. The aim of this fundamental in-vitro gene ex- After removal of the intron sequences by splic- periment – outside of the living mouse – was to ing, it becomes messenger RNA, mRNA, that prove that the muscle cells themselves can pro- moves to the ribosomes where it acts as the in- duce the therapeutic antisense oligribonucleo- formation transmitter for protein synthesis. Exon skipping with internally produced
For a human application, U7-snRNAs adapt- oligonucleotides: For a new method of skipping
ed to the individual mutation of the patient exons, the antisense oligoribonucleotides do not would have to be used, whose gene must then be have to be injected but are synthesized in the cell transported with a gene therapeutic vector into nucleus, where they are needed, after transfer of the cell nuclei of the muscle fibers. An alterna- their gene. The intron sequences are cut out from tive would be an ex-vivo transfer transporting the pre-mRNA in the cell nucleus by spliceo- the U7-snRNA genes into satellite cells or other somes. They are complex structures consisting of muscle stem cells and then injecting them into several proteins and small RNAs, small nuclear the blood stream or directly into the muscles. As = snRNAs, which recognize the exon-intron bor- the U7-snRNA genes are very short, this transfer ders and which join the exons after splicing pre- would probably be easier than the transfer of the cisely and without shifting the reading frame. cDNA for the entire or the shortened dystrophin Some of these very short RNAs are the U7- as is being tried with other methods (Weis, Bern; snRNAs. They bind to splice sites at special re- cognition sequences in the pre-mRNA, block the Homologous recombination: The point mu-
splicing of specific exons, and thus ensure that tation in the dystrophin gene of mdx mice could several proteins of different length are produced be repaired in 15 to 20 % of isolated myoblasts by one gene. The U7-snRNAs normally regulate by the addition of a DNA string of 603 base pairs the splicing processes of the mRNA of histones. whose sequence is the same as the sequence be- Histones are proteins necessary for packaging fore and after the mutation site of exon 23 of the mice but not containing the C-to-T exchange of For this new method, the U7-snRNAs were the mutation. Part of the new gene segment was genetically modified so that they no longer exchanged against the corresponding, the ho- bound to the histone pre-mRNA but only to the mologous, in exon 23. This short fragment ho- splice sites in the region of exon 23 of the pre- mologous recombination, SFHR, technique was mRNA of the mouse dystrophin. To achieve this, then applied to isolated myoblasts from a a gene coding for the modified U7-snRNA to- Duchenne patient with a deletion of exon 13 gether with a creatine kinase enhancer was packed into plasmids as vectors and then, in a Ignoring a premature stop codon by anti-
laboratory experiment, transferred into isolated biotics: About 5 % of Duchenne boys have a
myoblasts of the mouse. These cells then deve- point mutation in their dystrophin gene which loped in the culture dish into muscle cells. changed an amino acid code word into one of the In the nuclei of the transgenic myoblasts, the three stop codons, TGA, TAG and TAA, after transferred gene produced modified U7-snRNAs. which the synthesis of dystrophin stops prema- They had new recognition sequences and bound now in front and behind exon 23 of the dystro- Gentamicin is an antibiotic that causes the phin pre-mRNA to sites important for the splic- RNA translation mechanism in the ribosomes to ing process. Thus, these splice sites were block- ignore such a premature stop codon, i.e. to read ed and exon 23 was removed together with the through it. The normal stop codons, which are protected by a special three-dimensional struc- which showed, however, that under similar con- ture, will, however, be respected as before. In ditions after a treatment with gentamicin no new mdx-mice, up to 20 % of the normal amount of dystrophin could be detected (Karpati, Montreal; new and functional dystrophin has been obtained in this way. Gentamicin has the advantage of be- Two clinical studies with gentamicin have ing a well known drug whose use as a possible been performed on Duchenne boys but have also therapy for Duchenne muscular dystrophy would not led to new dystrophin. Possibly the 14-day not need long approval procedures (Sweeney, trial period was insufficient. Therefore, another and longer lasting clinical trial with 36 patients is In order to confirm these positive results, two now being performed (Mendell, Columbus). other studies with mdx mice were performed Replacement of dystrophin
Utrophin is a muscular protein present in small amounts also in Duchenne patients.
In larger quantities it could take over the function of dystrophin.

Upregulation of the utrophin gene: Utrophin is
a known drug, or some other chemical or a natu- a protein with a structure and function very simi- lar to dystrophin. In humans, its gene is located During the last years, synthetic chemistry has on chromosome 6, it has 75 exons and is about developed methods to automatically produce one million base pairs long. The utrophin protein thousands of partly unknown substances. Many is about 7 % shorter than dystrophin. It is present of these substances are being tested in the labo- in many body tissues, also in muscle, but there it ratory, also automatically, on cell cultures from is concentrated in regions where the motor ner- mdx mice for their ability to activate the gene of ves contact the muscle membrane. Before birth, luciferase, which is preceded by the two promot- the utrophin concentration in muscle is much ers of the utrophin gene. The light producing en- higher than afterwards. This protein, though it is zyme luciferase from fireflies is easier to detect present only in small amounts, makes the Du- than utrophin. Every hit, i.e., every substance chenne symptoms less severe than they would be which shows at least a low activity in these pre- if utrophin were also missing. In fact, mdx-mice liminary tests, is further modified, and then, all whose utrophin gene was knocked out experi- these similar substances are tested first on mus- mentally, which thus have neither dystrophin nor cle cell cultures, and, if they react positively, in utrophin, have Duchenne-like symptoms and die living mdx mice, too, to see whether they can early in contrast to “normal” mdx mice whose also upregulate the utrophin gene. Only after one or several convincingly active substances are found, would it be possible to start clinical stud- utrophin, if it is present in larger amounts, can ies with Duchenne patients in a few years (Da- replace dystrophin. The mice used were trans- genic mice who contained utrophin mini genes in Such an activator of the utrophin gene could, their germ line, introduced by a technique that if it is a small molecule, be applied via the blood cannot be used in humans. Other transgenic mice stream from where it would reach all the mus- were raised which produced utrophin only when cles. And the immune system would recognize they were given the antibiotic tetracyclin in their an additional amount of utrophin as a substance drinking water. The increased amount of utro- of its own because it is already present in small phin prevented the development of Duchenne amounts in Duchenne boys. Therefore, no im- symptoms, and this effect was more pronounced in newborn than in 10 or 30 day old mice. However, something that upregulates the For a possible Duchenne therapy, another utrophin gene, could also do the same with other strategy is followed, namely to increase the nor- genes. Thus, before such an activator is tested in mally low amount of utrophin by upregulation of children, it should be made certain that it does the activity of its gene. To achieve this, an acti- not produce any undesirable side effects. vating substance is needed, which could well be Other experiments to increase utrophin:
Arginine, therefore, cannot be used for a Du- The transfer of a shortened utrophin gene with chenne therapy without further investigations. --- adenoviruses into dystrophic dogs led to utrophin The small protein heregulin with which the mo- which could take over the function of the miss- tor nerves stimulate muscle development can ing dystrophin. --- Glucocorticoides, among also significantly alleviate the symptoms of mdx them prednisone, can upregulate the utrophin mice. --- One end of the utrophin mRNA is not gene to some extent. --- Such corticoides have translated into protein, but binds to structures at also been isolated from Chinese plant medicines the contact regions of the nerves, thus restricting which traditionally are used against muscular utrophin to these sites. If this binding could be dystrophy. --- Low grade chronic inflammation prevented by a drug, it might become possible to in muscles of mdx mice leads to a marked upre- distribute utrophin more evenly over the entire gulation of utrophin on the membrane outside of muscle membrane so that it can better replace the contacta with the motor nerves. --- The ami- dystrophin. --- Another form of utrophin, the no acid L-arginine can increase the amount of very similar B-utrophin was recently identified utrophin in mdx mice and alleviate significantly in blood vessels. But only the “normal” A-utro- the dystrophic symptoms. The enzyme nitric ox- phin is present in muscles and can partially com- ide synthase uses arginine for the production of pensate for dystrophin after upregulation of its the biologically active gas nitric oxide. But nitric oxide is also active in other biological processes. Other proteins
The mutations of the dystrophin gene and the absence of dystrophin
influence the activities of many other genes.

Activities of thousands of muscle genes: In or-
gy production in muscle cells were less active in der to measure simultaneously the activities of mice without dystrophin and utrophin, i.e. their very many genes in one tissue sample in a single muscles had an energy crisis which contributed experiment, gene arrays are used. As the sequen- to the degeneration of their dystrophic muscles. ces of practically all human genes are known, On the other hand, many genes necessary for the short segments of thousands of genes can be pro- development and repair of muscles were in- duced automatically and applied by a robot in a creased in their activities, in some cases by a certain pattern to a quartz chip a few square cen- hundred times, i.e., they were upregulated by the timeters in size. If biochemically produced DNA disease process. Similar results were obtained copies of the mRNAs of all active genes are app- lied as an analysis sample to the chip, light Further experiments showed that in mice ma- points appear at those chip sites where there are ny other genes were upregulated, genes which complementary DNA sequences of these active are involved in the development of surface struc- genes. The light intensity of these points is auto- tures, in the production of signal factors for pro- matically measured which then makes it possible tein synthesis, in the intensification of immune to determine which genes are active and to what reactions, and in other processes responsible for the muscle dystrophic symptoms of mdx mice. In With this expression profiling, several thou- Duchenne patients, these changes were less pro- sand genes in muscle samples were tested which nounced, and in transgenic mice with human came from normal and mdx mice, from healthy dystrophin, these gene activities were normal, and Duchenne boys, from transgenic mice which because they no longer had a muscular dystro- neither had their own dystrophin nor utrophin, and also from other mice which instead had hu- After these first results were obtained a few years ago, many more experiments with this new The results showed that the absence of dys- technique were performed to answer other ques- trophin causes the increase and the decrease of tions of Duchenne research. Their and future re- the activities of many muscle genes. A whole se- sults will help to understand the complex rela- ries of genes which are responsible for the ener- tionship between the many parts of the muscular architecture and its changes if one of its most im- velopment of muscular dystrophy (Ségalat, portant components, dystrophin, is absent. This will open new avenues for the development of a Integrins and synthrophins: Integrins are a
Duchenne therapy (van Ommen, Leiden; Kunkel, family of proteins which are located in the mus- Boston; Hoffman, Washington, and others). cle cell membrane. They are necessary for the A dystrophic worm: Caenorhabditis elegans
fusion of myoblasts to myotubes and the deve- is a 0.9 millimeter long transparent worm which lopment of myotubes to mature muscle cells. is used extensively by gene researchers because They also participate in the propagation of sig- all its 19,733 genes are known and also all its 959 body cells, 95 of which are muscle cells. Its In mice without dystrophin and utrophin, the muscles have a dystrophin similar to the dystro- amount of one of these integrins was increased phin of humans, which can also have mutations about twofold by gene transfer. This extended the life expectancy of the mice by threefold, and Individual genes were inactivated and also ac- their dystrophic symptoms ameliorated signifi- tivated so that the dystrophic symptoms could be studied which were caused by the missing or in- The five known syntrophins are proteins that creased gene activities. E.g., it could be shown mediate the interaction of signalling proteins that the upregulation of dystrobrevin, a very with the dystrophin and utrophin complexes at short form of dystrophin, slowed down the mus- the muscle cell membrane. Understanding these cle degeneration significantly. Further investiga- effects in more detail may have consequences for tions will contribute to the clarification of still a therapy with utrophin (Froehner, Seattle). unknown molecular relationships during the de- Pharmacological strategies
Corticosteroids and other drugs can ameliorate the symptoms
of Duchenne muscular dystrophy without curing the disease itself.
As long as the efforts to transfer a functional regulation of the myogenic precursor cells. Other dystrophin gene or to repair the damaged gene factors are also involved in optimizing muscle have not led to a cure, attempts are being made to at least alleviate the symptoms of Duchenne These facts led to the assumption that by dystrophy by a drug treatment. There has re- blocking the activity of myostatin, the muscles of Duchenne boys could be made larger or at least Myostatin: The “blue-white” Belgian cattle
their destruction reduced. Therefore, monoclonal have existed for about 200 years, they have 20 % antibodies were made, i.e., immune proteins that more muscle meat than normal animals. Six attach themselves very specifically only to myo- years ago, it was found that these cows have a statin and thus inactivate it. These antibodies deletion of 11 base pairs in the gene for the pro- were injected once a week under the diaphragm tein myostatin. Transgenic mice without a my- of mdx mice. After three months, the treated ostatin gene are two to three times as heavy as animals were 12 % heavier than control animals normal mice, not because they have more muscle without treatment because their muscle mass had cells, but because their muscle cells are much increased. In addition, they had a better muscle larger. Myostatin is a signal protein, a kind of function, they could better cling to a rotating hormone, it consists of 375 amino acids and is glass rod, their muscle degeneration had decreas- ed and the CK activities were practically normal. This protein is produced in the muscle cells Further experiments are now being performed and their precursor cells, after which the protein with mice. They have to be repeated with dystro- is modified: two thirds of the amino acid chain is phic dogs whose dystrophy is more similar to the removed, and two of the remaining chains with human disease than the dystrophy of the mice. 109 amino acids each form a double ring. This Only when the results are positive could clinical active myostatin inhibits the growth of the mus- cle cells by negatively influencing the genetic This treatment method would not be a cure of Duchenne muscular dystrophy because the gene- Other pharmacological experiments: Ex-
tic cause of the disease would not be eliminated. tracts of green tea in the food of mdx mice slow- But, compared with other methods, it would ed down the degeneration of some of their mus- have advantages: no immune or toxicity prob- cles, possibly because this tea contains anti-oxi- lems, no genetic risks by viruses, and easy manu- dant substances. --- Transgenic mice were raised facturing of the drug. Pharmaceutical companies which produced in their muscles the muscle insu- are already interested in this technique because lin-like growth factor 1 (mIGF1) in relatively increasing the muscle mass would also be im- large amounts. This increased muscle mass by up portant for older persons and people with other to 40 %, and fibrosis as well as muscle degen- muscle-degrading diseases (Khurana, Philadel- eration were decreased and the regeneration sig- nificantly improved. --- Leupeptin consists of Glucocorticoides: The related corticoides
three partly modified amino acids. Combined prednisone, prednisolone, and deflazacort delay with carnitine, it inhibits the enzyme calpain muscle degeneration, but the cause of this effect which destroys proteins in the muscle cells is not yet known. While part of their action likely when, as in Duchenne dystrophy, calcium enters involves their anti-inflammatory properties, in an uncontrolled way. In experiments with other mechanisms of action are also possible. By monkeys and mice, muscle degeneration was analyzing the activities of more than one thou- significantly slowed down by leupeptin. --- The sand genes in prednisone-treated mdx mice with concentration of the enzyme nitric oxide syn- the new micro array technique, it was found that thase in the dystrophin complex of mdx mice is about 5 % of the genes showed reduced or in- significantly reduced. Therefore, its product, the creased activities. Further analysis of the pattern biologically active gas nitric oxide, can no longer of gene expression induced by glucocorticoids fulfil its function. This contributes to muscle de- will help to understand the molecular mechanism generation. Transgenic mdx mice with the nor- of action of glucocorticoids in skeletal muscle. mal amount of nitric oxide synthase showed re- This might help to develop a more specific but duced dystrophic symptoms. --- In mdx mice, less toxic treatment with these drugs (Muntoni, the signal protein JNK1 is activated and this con- tributes to the degeneration of muscle cells. The Creatine can possibly also slow down muscle
injection of adenoviruses carrying the gene for degeneration. It is a natural compound which is the natural protein JIP1 inhibits the activity of eaten in large quantities by athletes for enhance- JNK1, and this reduces muscle degeneration. --- ment of performance. Creatine when bound to The protein galectin-1 participates in the proc- phosphoric acid provides energy not only for esses leading to new and regenerated muscle tis- muscle contraction but also for the removal of sue. Fibroblasts obtained from the skin of new- superfluous calcium, one of the causes of des- born mice develop into muscle cells with dystro- truction of muscle cells. Experiments with mdx phin if they grow in a cell culture which contains mice have shown that creatine supplementation galectin-1. Such fibroblasts could be easily col- can improve their disease symptoms, and this lected from a Duchenne patient, transformed into may provide a scientific basis for its use as sup- muscle cells with galectin-1, then genetically plementary therapy for Duchenne dystrophy modified to synthesize normal dystrophin, and (Wallimann, Zürich; Rüegg, Lausanne). finally transplanted back into the patient. Clinical studies with Duchenne patients
The first gene transfer trial and a new trial with myoblasts have begun.
Cortisteroids, creatine, and other chemical substances are studied.
A large study with prednisone together with cyclosporin is being prepared.

Clinical studies with Duchenne boys will be
phase I, will already take several years to prove more and more necessary in view of the increas- that the new treatment will not be accompanied ingly positive results with experimental animals. by unacceptable side effects. Only afterwards These studies with humans will have to be per- can further studies be started with sick children formed in several steps, of which the first one, to ascertain whether the treatment really im- proves or maintains the muscle force, phase II, and what the optimal dosage will be, phase III. A solution containing 0.2 mg of plasmids All these studies have to be performed doub- with 10 trillion (10 x 1012) copies of the dystro- le-blind, i.e., only about half of the patients re- phin gene was injected into one muscle of the ceives the substance to be tested whereas the forearm of the first three patients. This is a very other half receives an inactive compound, a pla- small amount of genetic material compared to cebo. And neither the patients nor the researchers similar experiments in animals. The next three are allowed to know which patient belongs to patients received one dose of 0.6 mg and the last which group before the trial is completed, the three two doses of 0.6 mg two weeks apart. The code is broken and the results are analyzed. safety of the patients was the main concern, These studies and the approval procedures are therefore, any one patient was treated only when time consuming, they take many years, and are it was certain that the previous one treated did not show any signs of immune intolerance. Transfer of the dystrophin gene with plas-
Three weeks after the injections, the treated mids: The first phase of the first gene transfer
muscle area of about 0.5 cubic centimeters was experiment with Duchenne patients has been extracted by biopsy and checked for the presence completed at the beginning of 2003 and the re- of dystrophin. In three out of six boys in the first sults reported in June 2003. The biotechnology two groups and in all three boys in the third company Transgène in Strasbourg together with group, new dystrophin appeared in less than 1 % the French muscular dystrophy association AFM to more than 25 % of the muscle fibers around started to prepare this gene therapy approach in the injection sites. There were no signs of an im- 1995. The permission for this first human trial mune reaction, neither to the plasmid nor to the was given by the French authorities in November newly produced dystrophin. This answered the 1999, and the first injections of the vectors were question of a phase-I study: Gene transfer with performed in September 2000 at the Hôpital de naked DNA is a safe procedure. It could, after amplification, become a therapeutic method be- The 9 participating boys were all older than cause it is known from animal experiments that 15 years so that they could give their informed dystrophin production in about 20 % of muscle consent. They did not derive any clinical benefit fibers would improve muscle function signifi- from this treatment, it was not yet a therapy. After several gene transfer methods were test- The French scientists are now working with ed on dystrophic mice and dogs, it was decided the team of Jon Wolff in Madison in the United to use the entire cDNA of the gene for the full- States, who injected similar plasmid construc- length dystrophin placed in a plasmid as vector tions with genes of a marker protein into the together with a strong promoter from a virus. blood stream of limbs of rats, dogs, and monkeys Plasmids have the advantage of not containing under pressure. Afterwards, up to 40 % of the any protein and thus should not cause an immune muscle fibers contained the transferred marker reaction. The therapeutic gene to be transported has no protein either, it is pure or naked DNA. The next step will be to apply this arterial de- In further preliminary experiments with mus- livery procedure in Duchenne boys, probably in cle cell cultures and mice, it was shown that this a clinical trial starting in 2004. At that stage, it is vector construction led to the appearance of new planned to treat, again for safety reasons, only a dystrophin at its correct place underneath the small foot muscle. Should the results be positive, muscle cell membrane of the animals, that it re- this method will be tried on entire arm or leg stored the dystrophin-glycoprotein complex, and muscles. This could be planned for 2006. that it prolonged the life of the cells. Afterwards, respiratory and cardiac muscles The aim of this clinical study with Duchenne could be targeted. For safety reasons, it is impos- patients was to show that the procedure is safe, sible to proceed faster, because it would be a ca- i.e., that is does not lead to an immune reaction tastrophe should severe side effects or any other or an inflammation, and that new and normal undesired event happen which would lead to the dystrophin appears at the correct places in those interruption of this and other gene therapy ex- fibers of the muscle which had received the Clinical studies with myoblasts (myoblast
maintain the muscle force for at least two to transfer): At the beginning of 2003, a clinical three years and, in isolated cases, prolong walk- phase-I trial with 5 to 15 year old Duchenne ing ability until about the 14th year. The most boys with deletions has started in Québec City in important side effect of prednisone was weight Canada. It should answer the question whether gain in about 20 % of the patients. With de- the transfer of normal myoblasts under modified flazacort, slight cataracts, clouding of the eye conditions is safe, i.e., that it does not create an lens, were more frequent than with prednisone. immune rejection or inflammation, and whether Both drugs had a growth retarding effect, other new dystrophin appears after the treatment. side effects were insignificant. After stopping the The difference with regard to the unsuccess- medication, muscle degeneration and normal ful experiments performed in 1990 is that the much more effective immune inhibitor FK506 The results of the study do not yet allow a de- (Tacrolimus) A is used instead of cyclosporin, cision on the best time to begin the treatment, that not, as before, 60 to 90 million cells are in- e.g. before five years of age. Children who wish jected into the entire biceps muscle, but 30 mil- to begin this treatment should do this in the set- lion cells into the foot muscle tibialis anterior by ting of a well documented study in order to ob- multiple applications at a distance of one milli- tain more information and, because of the side meter from each other into a muscle volume of effects, to guarantee the necessary controls. The only one cubic centimeter. An improvement of study in Germany is continued as an open trial the muscle function is not expected. As in the with long-term documentation which includes French trial, the participants will have no thera- data for some patients who were treated for more peutic benefit from these injections. than seven years (Reitter, Mainz). One month after the treatment and after a bi- Prednisolone: In the United Kingdom, a
opsy it will be determined whether normal dys- large long-term trial had been prepared which trophin DNA, mRNA and protein have appeared should have tested whether prednisolone is able and whether there were any immune reactions. to prolong walking ability and to improve life Three patients have been treated until July 2003, quality. An intermittent treatment, 10 days with the entire trial should be finished before the end and 10 days without medication had been plan- ned. Because financing could not be obtained, it If the results are positive, the trial will conti- is now only possible to conduct an open study, nue with a phase-II study, during which myo- not double-blind, with the aim of documenting blasts will be transferred into the entire biceps efficacy and side effects (Muntoni, London). muscle. Then, during the following two years, Prednisone combined with cyclosporin A:
the muscle force will be measured which, as is In Germany, a clinical study is being prepared to hoped, will have possibly increased or at least start at the end of 2003 which should answer two remained unchanged (Trembley, Québec City). questions: Can cyclosporin A alone increase Prednisone and deflazacort: Sixteen clinical
muscle force over a short time? And can a com- studies worldwide had proven the ability of glu- bination of cyclosporin A and prednisone better cocorticoides, cortisone derivatives, especially of reduce the loss of force over the long term than prednisone, to maintain the muscle force of Du- prednisone alone, when prednisone is given in- chenne boys. A few years ago, there were indi- termittently, i.e., in a cycle of 10 days with and cations that the new corticoid deflazacort, which 10 days without therapy? As immune processes is related to prednisone, would act similarly but play a role in Duchenne muscular dystrophy, ear- lier experiments have shown that immunsuppres- From 1992 to 1997, a study with the partici- sant drugs such as cyclosporin A may delay mus- pation of 14 German muscle centers was per- formed in which the muscle maintaining effects In order to obtain reliable results, at least 150 of these two drugs were compared with the well Duchenne patients should participate who are documented natural history of the disease. The unequivically diagnosed, who are older than six doses were 0.75 mg per kg body weight and day years, and who can still walk alone for 50 me- for prednisone and 0.9 mg per kg and day for de- ters. During the first phase lasting three months, flazacort. The result was that both drugs can all children will receive, in a double-blind proce- dure 3.5 to 4 mg/kg/day cyclosporin A alone or pounds had been tested in a large screening ex- galactose, milk sugar, as placebo. During the se- cond phase lasting 12 months, all children will Albuterol, an asthma drug, had shown a sig-
receive in addition 0.75 mg/kg/day prednisone nificant increase of muscle strength (about 8 %) for 10 days followed by 10 days without predni- and very few side effects in a preliminary trial sone. Eight German muscle centers will perform with 10 Duchenne boys. In August 2002, a doub- this trial (Korinthenberg, Freiburg). le blind study has started with 25 – 30 boys to Creatine: A double-blind study with 8 Du-
chenne, 10 Becker, and 18 patients with other A trial with coenzyme Q10 started in Sep-
muscle diseases showed after 8 weeks with daily tember 2001 with 15 boys which also regularly doses of 5 grams creatine monohydrate for chil- receive deflazacort or prednisone. Another trial dren and 10 grams for adults a slight but signifi- is planned with patients in wheelchairs. cant beneficial short-term effect on muscle Oxatomide, an antihistamine, is being tested
strength and performance without any side ef- in a 9 months trial with 15 Duchenne boys. fects. More clinical studies have to be made be- Pentoxifylline interferes with the immune
fore creatine can be recommended as a long-term system and thus reduces inflammation and fibro- muscle preserving medication for Duchenne sis. A trial started in February 2003 and will last In Canada, a trial to determine the effect of A trial lasting 15 months has started in Janu- creatine has started with 40 Duchenne boys. --- ary 2003 to compare the positive and negative In Belgium, creatine was tested for three months effects of prednisone when it is given every day
on 12 Duchenne and 3 Becker patients with the or only on two days each week at a higher dos- result of a slightly increased muscle force. Oxandrolone, an anabolic steroid sometimes
A trial with 54 Duchenne boys was performed used by athletes, was tried in a study with 51 for 6 months ending in March 2003 in which ei- Duchenne boys. However, the small improve- ther creatine or glutamine was tested in a doub-
ment of muscle strength found does not justify le blind study. The results are being evaluated. its use as a drug instead of prednisone or defla- Trials with three other substances, taurine,
carnitine, and nicotinic acid, are planned.
International clinical tests: The Cooperative
For the documentation and supervision of International Neuromuscular Research Group, these studies, standardized control methods have CINRG, in Washington, a cooperation of labora- been developed to measure not only muscle tories in the US, in Canada, Belgium, Argentina, functions but also other parameters such as the Australia, and India, organizes clinical trials on quality of life. Some of these methods are being Duchenne boys with substances some of which modified so that they can be also used for very have been selected from 45 substances which young and older patients (Escolar, Washington). had shown positive results when many com- When will there be a therapy?
Duchenne muscular dystrophy has always been phin gene, identified (Kunkel, Boston) and short- with man and all animals with skeletal muscles. ly afterwards the protein dystrophin characteriz- Its clinical symptoms were described quite cor- ed (Hoffman, Washington), which is missing in rectly for the first time in 1851 by the English Duchenne boys. The fast pace of genetic re- doctor Edward Meryon. But, it got its name after search gave rise to the hope that it would soon be the French physician Duchenne de Boulogne, possible to replace or repair the gene and thus who, in 1861, described not only its symptoms but also the muscular changes, its histology. However, this optimism was premature. The From its mode of inheritance, it was known at first studies in 1991 with the myoblast transfer the beginning of the 20th century that a defect on showed that this technique, which looked prom- the X chromosome is responsible for the disease. ising in mice, was ineffective in Duchenne boys. But only in 1986 was the gene itself, the dystro- Now, 17 years after the detection of the gene, there is still no therapy for Duchenne muscular and many more obtained before it is possible to

Source: http://www.duchenne-information.eu/research.pdf