Muscular Dystrophy (towards a cure).

Now the gene for DMD/BMD and some of the other MDs have been isolated and sequences, and we know their approximate functions. How can we design rational strategies to halt and perhaps even reverse the course of these diseases? Ideally, we would wish to have a good animal model as the early trials may well be uncomfortable. However, we may have to do without attempting instead to go straight to clinical trials with human volunteers.

The MDX mouse, is it a sufficient model for DMD/BMD? Despite the fact that the MDX mouse lacks dystrophin it seems remarkably unaffected. MDX mice have similar life span expectancy to wild type mice (around 2 years), and do not show any clinical signs. The two papers given at the end of the first dystrophin lecture (Grady et al 1997 and Deconinck et al, 1997) conclude that both utrophin and dystrophin have to be ablated in mice in order to recreate the pathologies seen in DMD/BMD patients who of course only have deficiencies in the dystrophin gene. This is a major . Although these and other experiments show that the MDX mouse is not a good model for the human disease and that the double K/O mice are also not ideal, they show that utrophin can substitute for dystrophin at least in the mouse. This at least is encouraging!

Can dystrophin be introduced to the muscles of DMD patients? The introduction of dystrophin into the muscles of patients who lack parts of or the whole of dystrophin is very likely to result in the patient producing antibodies to this protein as it will be perceived as being “foreign”. It may be possible to damp down the patients immune system in the same way that is routine after transplantation surgery, but this is not ideal. Another problem is that the cDNA of the full length dystrophin would be very long, a way around this is to try the “mini dystrophin” cDNA based on the gene product that a very mild BMD patient expressed (England et al, ).

Figure 11. Utrophin is expressed at the myotendinous junction and the neuromuscular junction in adult human.

Figure 10 Utrophin is structurally similar to dystrophin, but is two helical

repeats shorter

Figure 12

Utrophin, a substitute for dystrophin. As utrophin is so similar in structure and function to dystrophin many groups have concluded that some of the symptoms of DMD/BMD may be reduced if one were able to persuade muscle cells to produce utrophin. The studies with the MDX mice quite clearly show that utrophin does compensate for the lack of dystrophin in the mouse. Does this necessarily mean that the same would follow for humans? There are indications that this is so. It has frequently been noted that even in the most severely affected DMD patient that the extraocular muscles (around the eye, See Fig 12) are spared (Karpati & Carpenter, 1986). These muscles are rather special in that they express unusual myosin II isoforms and in that they express utrophin in addition to dystrophin. It seems that the extraoccular muscles are devoid of DMD pathologies because of the presence of utrophin as mice lacking utrophin and dystrophin show degenerationeven in the extraocular muscles (Porter et al, 1998). Usually, utrophin expression in skeletal muscles is restricted to the myotendinous junction and the neuromuscular junction (Fig 11). This is also the case in mouse, but here utrophin is found in skeletal muscle in the embryo, and in the MDX mouse where muscle regenerates. It is tempting to speculate that if one could artificially express utrophin in the muscle of DMD/BMD patients that this may substitute for dystrophin to some extend and alleviate the symptoms of the disease.

How do you get Utrophin cDNAs into muscle cells? Muscle cells have an usual ability to take up naked DNA and shuttle it into their nuclei. This of course led some to experiment with the simple injection of dystrophin cDNA constructs directly into muscle. However, the earlier optimism was curtailed as it soon became clear that the efficiency of this process was nowhere near sufficient.

Figure 13. The use of the "adeno-associated" virus (AAV) to introduce a working copy of the sarcoglycn gene to the foot muscle of a LGMD patient as an early trial of the technique. (adapted from the MDA web site http://mdausa.org/home.html )

Figure 11

The first human trial for a genetic treatment of a Muscular Dystrophy. The first treatment for LGMD was started on the 2^nd Sept 1999. Donavon Decker, a 36-year-old air traffic controller became the first person to receive a gene therapy injection for has a sacroglycan deficiency LGMD. The sacroglycan cDNA was inserted in a construct containing muscle-specific promotors in a "adeno-associated" virus (AAV) injected a muscle on the top of the foot. AAV is not pathogenic for humans but the replicative machinery was removed in the strain used for this study to be safe. The AAV carrying the sarcoglycan cDNA was injected into a muscle in one foot while the same muscle of the other foot received a sham injection. Biopsies will be taken from the muscle of both feet to compare the condition of the two muscles after six weeks. Integration of the viral DNA into the genomic DNA will be checked in addition to expression of the sarcoglycan construct. Evidence for the appearance of the previously absent sacroglycan (and the other sacroglycans, whose expression is also dramatically reduced in this LGMD) will be monitored. Damage to the muscle cells will also be investigated. The patient will be monitored closely for any adverse reactions, including immune response or other unwanted response to the virus used to carry the genes, to the genes themselves, or to the protein molecules produced by the genes. In anticipation of all going well with this single patient trial the MDA are gearing up for a full scale trial. The results of the trial will be published by the MDA on their web-site at:- http://mdausa.org/home.html

Although the AAV is known not to cause serious immune response it is not certain whether this comparatively massive dose will cause some problems. If the sacroglycan cDNA is expressed in the muscle, it is possible that the pateints immune system will see this as foreign. There are problems to be overcome, but this is clearly a promising development.

Can the same process work for DMD/BMD?

The main difference between DMD/BMD and sacroglycan LGMD is size. The dystrophin cDNA is huge and requires a different viral delivery system than that being used in the LGMD trials. Unfortunately, the best available virus large enough to contain the dystrophin gene, the adenovirus,is immunogenic. In the mouse it has been possible to introduce truncated dystrophin constructs successfully, and these produce proteins which restored the DGC (Yuasa et al, 1998) To try to get around this problem, researchers have been working on a new generation of "stealth" adenovirus carriers that few of the original viral genes and are designed to attract as little attention as possible from the body's immune system. These vectors are known as "gutted" vectors. In addition to prolems related to the antigenicity of the viruses, the dystrophin protein lacking in DMD patients would have to be present at the sarcolemma at the normal concentration of 2% total membrane protein, and being “new” would also attract attention from the immune system. One possible solution is to use the mini-dystrophin gene. This mini-gene was discovered in a mild BDM patient (still able to walk at age 60) (England, et al 1990), however, it is known that the same mutation can affect different people in very different ways and it is possible that the same massive deletion may in other patients be dissasterous. As mentioned previously, a better strategy would be to produce full length utrophin in the muscles of DMD/BMD patients.

Muscle cell culture as an alternative strategy

Several studies have concluded that it is possible to excise myoblasts from mice culture, amplify their numbers by tissue culture and reintroduce the cells to the same animal. These myoblasts produce muscle tissue with the correct extracellular matrix support The tissue even becomes innervated (Irintchev et al, 1998), this opens up the possiblity of repoulating diseased muscle with genetically transformed myoblasts. However, a number of tumours are produced by this process. More hopeful is the observation that there seems to be a progenitor muscle cell population present in the bone marrow that circulates out of the bone marrow and into peripheral muscle via the circulation. This offers the intriguing possibility of using this system to re-populate the degenerating muscle with cells carrying the correct or corrected genes (Partridge, 1998).

Conclusions

The MDX mouse is not a suitable model system for DMD/BMD and the MDX:utrn^-/- mice may not be much better. Indeed since mouse muscle cell up-regulate utrophin enough to continue regeneration, the mouse may not be a good choice of animal at all. Dystrophic cats, and dogs are available, but are not favourites for ethical reasosns. Dystrophin is so large that it is likely to cause an immune reaction, the mini-dystrophin gene may not be useful in all genetic backgrounds. The brightest possibility seems at this stage to increase utrophin expression in the muscle cells. This may be best achieved by introducing viruses carrying a suitable cDNA, or perhaps by the introduction of muscle cell pro-generators transformed with utrophin encoding cDNAs. For the long term strategy, it may be advisable to offer genetic councelling to those whose backgrounds pre-dispose them for some of the recessive MDs along with suitable genetic tests, and for those DMD/BMD patients successfully treated by genetic intervention.

References

Deconinck , A.E. et al, (1997). Utrophin-Dystrophin deficient mice as amodel for Duchenne muscular dystrophy. Cell 90;717-727.

England, S.B. et al (1990) Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 343; 180-182.

Grady, R.M. et al (1997). Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin : a model for Duchenne muscular dystrophy. Cell 90; 729-738.

Irintchev, et al, (1998). Ectopic skeletal muscles derived from myoblasts implanted under the skin. J.Cell Sci. 111; 3287-3297.

Karpati, G. & Carpenter, S. (1986). Small-calibre skeletal muscle fibers do not suffer deleterious consequences of dystrophic gene expression. Am.J.Med.Genet. 25; 653-658.

Partidge T(1998) Fantastic voyage of muscle progenitor cells. Nature Medicine 4; 554-555.

Porter, J.D. et al, (1998). The sparing of extracolar muscle in dystrophinopathy is lost in mice lacking utrophin and dystrophin. J.Cell Sci. 111; 1801-1811.

Rafael, J.A., Tinsley, J.M., Potter, A.C., Deconinck, A.E. & Davies, K.E. (1998). Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice. Nature Genetics 19; 79-82.

Winder, S.J. (1997). The membrane-cytoskeleton interface: the role of dystrophin and utrophin. J.Muscle Res. Cell Mot. 18; 617-629.

Yuasa, Y. et al, (1998) Effective restoration of dystrophin-associated proteins invivo by adenovirus-mediated transfer of truncated dystrophin cDNAs. FEBS letters, 425 329-336.