Compositions for and methods of treating muscle degeneration and weakness

Compositions for and methods of treating muscle weakness and degeneration are described. Such compositions include myogenic cells which are administered by the described methods to one or more affected muscles.

The invention described herein was made in the course of or under grants 
from the National Institute of Health. 
BACKGROUND OF THE INVENTION 
This invention pretains to compositions for and methods of treating muscle 
degeneration and weakness. More particularly, the present invention 
relates to myogenic cells and methods of using such cells in the treatment 
of muscle degeneration and weakness. 
Progressive degeneration and weakness of skeletal muscles are hallmarks of 
the forty human neuromuscular diseases affecting motoneurones, peripheral 
nerves and/or muscles. Most of these diseases are fatal, and all are 
crippling. There is no known cure or effective treatment. These diseases 
include motoneurone disorders, such as Amyotrophic Lateral Sclerosis (ALS) 
and neuromuscular junction disorders, such as Myasthenia Gravis and 
Eaton-Lambert Syndrome. Also included are the twelve hereditary muscular 
dystrophies, predominantly muscle diseases, affecting over 200,000 
Americans. In the muscular dystrophies, dystrophic cells degenerate 
because of the lack of normal genome. 
Muscular dystrophy in the mouse is characterized by progressive 
degeneration of skeletal muscles in the hindlimbs and in the chest wall. 
Dystrophic symptoms first appear at 20 to 30 days after birth and consist 
of sporadic flexion and flaccid extension of the hindlimbs. Occasionally, 
the dystrophic mouse walks with duck feet (See for example, Michelson et 
al., Proc. Nat. Acad. Sci., 41: 10798, (1955) and Meier et al., Life Sci., 
9: 137, (1970)). A number of approaches have been employed by researchers 
in the field to study and develop methods to treat the muscular 
dystrophies and other neuromuscular disorders. 
In the case of the hereditary neuromuscular disorders, one approach to 
correct the genetic disease is to correct the abnormal gene itself. 
However, before gene therapy can be used to treat hereditary myopathies, 
the defective genes and their expression have to be determined. Although 
identification of the dystrophic genes and their primary protein 
abnormalities has been attempted by some workers, thus far, attempts at 
identification have not been completely successful. (See e.g., Monaco et 
al., Nature 323: 646-650, 1986; Brown et al., Hum. Genet. 71: 62-74, 
1985). Furthermore, before gene therapy can be used to treat hereditary 
myopathies, the problems of nonspecific gene integration, replacement, 
targeting, regulation and expression also have to be overcome. The high 
spontaneous mutation rate also complicates the process of identification 
and prevention. (See e.g., Epstein et al., Am Sci 65: 703-711, 1977.) When 
normal and dystrophic tissues are compared, the dystrophy-specific protein 
difference is often masked by the concomitant presence of 
individual-specific protein differences (see, e.g., Komi et al., Acta. 
Physiol. Scand. 100:385-392, 1977) and secondary degenerative changes 
(See, e.g., Dolan et al., Exp. Neurol. 47:105-117, 1975). In situations 
where the primary protein abnormality is not known, any trial of drugs to 
treat the disease will necessarily be arbitrary and its success 
coincidentally limited. (See, e.g., Bhargava et al., Exp. Neurol. 
55:583-602, 1977.) 
In Duchenne muscular dystrophy, carrier detection and prenatal diagnosis 
seek prevention rather than cure. See, e.g., Bechmann, Isr. J. Med Sci 
13:102-106, 1977. These are inadequate measures, because not all 
sex-linked carriers--inasmuch as they are phenotypically normal--are 
exposed to the diagnostic tests. There are also the legal, religious, 
emotional and financial considerations involved in inducing an abortion. 
Various studies have been carried out in attempts to develop methods to 
treat neuromuscular disease. 
In one reported approach, mouse muscle mince transplants studies were 
conducted on normal and dystrophic littermates (Law, Exp. Neurol., 60:231, 
1978). In another study, it is reported that near-normal contractile 
properties were produced in adult dystrophic mouse muscle by grafting a 
muscle of a newborn normal mouse into a recipient muscle of a dystrophic 
mouse (Law et al., Muscle & Nerve, 2:356, 1979). It is also been reported 
that mesenchyme transplantation can improve the structure and function of 
dystrophic mouse muscle as demonstrated by histological, 
electrophysiological and mechanophysiological studies (Law, Muscle & 
Nerve, 5:619, 1982). 
Watt et al., Muscle and Nerve, 741-749, Nov/Dec, 1984, report the injection 
of normal myoblasts into apparently abnormal muscle of strain mdx mice, 
but do not report any improvement in muscle function, The mdx mice do not 
exhibit any muscle weakness. Myoabnormality of central nucleation heals 
itself with age. 
It has further been reported that injections of normal myoblasts into 
growing dystrophic mouse solei improved the structure and function of the 
solei (Law and Goodwin, Fifth Biennial Forum Regeneration Abst, 1985, page 
18; Law and Goodwin, Soc. Neurosi. Abst. 11:212, 1985; and Law and 
Goodwin, IV International Congress on Neuromuscular diseases, Abst. 9, 
1986). Improved muscle function was determined only by 
electrophysiological and mechanophysiological studies. In such studies, 
only one muscle, the soleus, rather than all major muscle groups were 
tested. The soleus, containing many red fibers that are slow twitching, is 
unique and different from other muscles in the body that are composed of 
fast twitching fibers. 
Various attemps have been made to provide treatments for neuromuscular 
disorders. However, none have achieved recovery of muscle function, 
locomotive pattern and respiratory function in a host affected with muscle 
degeneraion and weakness. Thus, compositions and methods of treating such 
disorders are being sought. 
SUMMARY OF THE INVENTION 
The present invention provides, for the first time, compositions for and 
methods of successfully treating muscle degeneration and weakness. The 
present invention also teaches the use of cloned cells for the successful 
in vivo treatment of such disorders. 
Accordly, the present invention provides a method of treating muscle 
degeneration and weakness in a host which comprises administering a 
treatment effective amount of myogenic cells to at least one myopathic 
muscle of the host. 
Although any myogenic cell may be used in the practice of the present 
invention, preferred cells include myoblasts, myotube cells and young 
muscle fiber cells. These myogenic cells may be cultured or cloned. They 
may further be histocompatible or histoincompatible with the recipient. 
Thus, the present invention provides compositions for and methods of 
treating muscle degeneration and weakness which are expected to enhance 
the function and quality of life of hosts who suffer such disorders.

With respect to FIG. 5 all cross-sections are all of the same 
magnification. Modified Gomori trichrome stain. Bar=400 um. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides, for the first time, compositions for and 
methods of successfully treating disorders such as muscle degeneration and 
weakness, for example, that accompanying neuromuscular disease. It was 
unexpectedly found that the claimed compositions and methods may be used 
to dramatically improve the muscle function, locomotive pattern and 
respiratory function of a host suffereing such disorders. Such treatment 
has not heretofore been achieved. It is also believed that the present 
invention demonstrates the first use of clonal cells for the successful in 
vivo treatment of such disorders. 
The claimed compositons and methods will be illustrated for the treatment 
of mice having hereditary muscular dystrophy. However, other mammals and 
other neuromuscular diseases and muscle degeneration and weakness and may 
be treated by the inventive compositions and methods. 
Dystrophic cells degenerate because of the lack of the normal genome, and 
it has surprisingly been found that the claimed compositions and methods 
may be used to incorporate the normal genome into dystrophic muscle to 
dramatically improve the function of the muscle. 
Two mechanisms are thought to be responsible for the incorporation of the 
normal genome in such cases: 
1. Surviving donor myoblasts develop into normal myofibers and replace the 
degenerative tissue; and 
2. Normal myoblasts fuse with dystrophic cells to form genetically mosiac 
myofibers of normal phenotype. 
Because the claimed compositions and methods are based on developmental 
processes universal to all mammals, it is expected to have broad clinical 
applications and to minimize problems relating to specificity of 
integration, complementation, regulation, and expresson of the normal 
genome inserted should be minimized. 
In the treatment of hereditary neuromuscular diseases by use of the claimed 
invention, it is not necessary to know which gene(s) is responsible for 
the disease. Furthermore, the administration of genetically normal 
myogenic cells directly into the dystrophic muscle eliminates the 
uncertainty of tissue targeting encountered with gene therapy. 
It is believed that any myogenic cell may be used in the practice of the 
present invention. However, preferred myogenic cells include myoblasts, 
myotube cells and young muscle fiber cells. Such myogenic cells may be 
either cultured or cloned. The myogenic cells may further be 
histocompatible or histoincompatible with the recipient. 
Myogenic cells may be cultured by a variety of methods known to those 
skilled in the art to produce a sufficient quantity of cells for use in 
the claimed invention. One such method is described by Law and Goodwin, 
Muscle and Nerve, 1988, In Press. 
Myogenic cells for use in the present invention may also be produced by 
cloning methods known to those skilled in the art. Whereas both cultured 
and cloned myogenic cells can provide a virtually unlimited supply of 
cells, cloned myogenic cells offer advantages over cultured myogenic cells 
in that cells having superior developmental characteristics may be 
selected and propagated for the practice of the present invention. Another 
advantage is that cloned myogenic cells, e.g., myoblasts, can be readily 
prepared which are essentially free from other cell types. In contrast, 
cultured myoblasts derived from mesenchyme comprise about 80% myoblasts 
and 20% fibroblasts and may contain other cell types and components. In 
some instances, it has been found that fibroblasts interfere with the 
practice of the claimed invention and cause detrimental effects. (See, Law 
and Goodwin, 1988, supra.) The use of clonal cell transplants to treat 
muscle degeneration and weakness and neuromuscular diseases has not been 
reported. Furthermore, cell clones are physiologically different from 
organs that are used in heart, lung, kidney and liver transplants. 
In order to reduce immunological rejection problems, myogenic cells may be 
cultured or cloned from muscle biopsies of normal parents or siblings of 
the dystrophic patients to minimize immunologic reaction (Hauschka et al., 
In Rowland LP (ed): Pathogenesis of Human Muscular Dystrophies Ex. Med., 
p. 835, Amsterdam, (1977)). If a host is diagnosed as having a hereditary 
neuromuscular disease at an early enough age, i.e., when cells are very 
young and regular, cell biopsies of such cells may be taken and maintained 
for later culturing or cloning for use in accordance with the present 
invention. Cloning further removes the more active antigenic factors such 
as leukocytes, (Lafferty et al., Transplant Proc., 8:349, (1976) and 
Lafferty et al., Ann. Rev. Immunol., 1:143, (1983)) and can be used to 
mass-produce the myoblasts (Feder et al., Sci. Amer., 248:36 (1983)). 
Although it is desirable to use histrocompatable cells in the practice of 
the present invention, it has been found that it is not necessary. 
Histoincompatible cloned cells were unexpectedly found to dramatically 
improve muscle function in living hosts. 
In accordance with the present invention, the myogenic cells are injected 
into one or more of the muscles of the host with a neuromuscular disorder, 
or in the case of a host with a hereditary neuromuscular disease, into a 
presumably pre-myopathic muscle. As used herein, "presumptively myopathic" 
means that a host has tested positive for a hereditary neuromuscular 
disease but does not demonstrate any apparent symptoms or pathology of the 
disease. The number and type of muscles selected for administration of the 
compositions of the claimed invention will depend upon the severity of the 
condition being treated and will ultimately be decided by the attending 
physician or veterinarian. 
The present invention teaches that administration of an immunosuppressant 
to a host allows histoincompatible clones of normal myogenic cells 
administered to the host to survive and develop in the skeletal muscles, 
thereby greatly improving muscle structure and function, and preventing or 
reducing muscle weakness, a primary cause of crippling and respiratory 
failure in hereditary muscular dystrophies. The immunosuppressant 
cyclosporin-A (CsA, Sandoz) was used, enabling clones of histoincompatible 
normal myoblasts to survive, develop, and to improve the structure and 
function of the dystrophic host muscles. However, other immunosupressants 
which are or may become known to those skilled in the art will find 
application in the present invention. 
The demonstration that CsA administration permits cloned normal myoblasts 
to survive and develop in histoincompatible hosts indicates that clonal 
cell lines of superior myoblasts can be established, selected against 
tumorigenicity, and stored in cell "banks" ready for injection. 
The dystrophic mouse is used as an animal model of hereditary muscle 
degeneration and weakening. Dystrophic mice and control normal mice were 
treated with the immunosuppressant CsA prior to adminstration of cloned 
myoblast cells to various muscles as described below in the Examples. 
Injection of histocompatible normal myoblast clones into dystrophic 
muscles improved the structure and function of the muscles to almost 
normal. Immunosuppression of the C57BJ/6J-dy.sup.2J dy.sup.2J hosts was by 
way of daily subcutaneous injection of CsA. 
Injected dystrophic muscles exhibited greater cross-sectional area, total 
fiber number, wet weight, and twitch and tetanus tensions six months 
postoperatively. Fiber typing was more defined and they contained more 
normal-appearing and less abnormal-appearing fibers than non-injected 
controls. 
Eleven out of nineteen mice that received myoblasts injections on both 
sides of the body showed such behavioral improvement that their locomotive 
patterns were indistinguishable from normal mice. Using dimeric isozymes 
as genotype markers for host and donor cells, the demonstration of 
parental and hybrid isozymes inside the injected muscles substantiated the 
survival and development of donor myoblasts into normal myofibers, and the 
fusion of normal myoblasts with dystrophic satellite cells to form 
genetically mosaic myofibers. 
The amount of CsA necessary to accomplish effective immunosuppression may 
be determined by methods known to those skilled in the art. Successful 
usage of CsA on mice has been reported with subcutaneous injection daily 
at 50 mg/kg body weight with a stock solution of 15 mg/ml, (Kunki et al., 
J. Immunol., 125:2526, 1980; and Klaus, et al., Transplantation, 31:266, 
1981 through the use of doubling, (Watt et al., Clinl. Exp. Immunol., 
55:419, 1984 and Watt et al., Transplantation, 31:255, 1981) or halfing 
(Gulati et al., Exp. Neurol., 77: 378, (1982)) of the dosage has also been 
reported. 
Before carrying out the work described above, a study was conducted to 
determine CsA toxicity on clonal myoblasts of the mouse. The myoblasts 
were cultured at CsA concentration of 0, 0.25, 7.50, 25, 75, 250, 562.5 or 
750 ug/ml culture medium. It was found that myoblasts survived and fused 
at CsA concentrations of 25 ug/ml or lower and that they degenerated at 75 
ug/ml or higher. Results of this study are illustrated in FIG. 1. FIG. 1A 
shows G8 myoblasts subcultures that had undergone over twenty serial 
passages, and were originated from Swiss Webster mose hindlimb muscles, 
(Christian et al., infra.) FIG. 1B shows myotube formation at 50 hours 
after plating and FIG. 1C shows myotube formation of 50 hours in the 
presence of CsA. FIG. 1D shows degenerative myoblasts 50 hours after 
plating in the presence of CsA at 75 ug/ml. In FIG. 1: Phase Contrast; 
Bar=50 um. 
Cloned myogenic cells may be administered as taught by the present 
invention for research purposes or may be administered therapeutically to 
mammals, including humans. 
The methods of the claimed invention can be used to administer myogenic 
cells ("the active ingredient") for the in vivo treatment of mammalian 
species by physicians and/or veterinarians. The amount of said active 
ingredient will, of course, depend upon the severity of the condition 
being treated, the route of administration chosen and the activity or 
potency of the active ingredient, and ultimately will be decided by the 
attending physician or veterinarian. Such amount of active ingredient as 
determined by the attending physician or veterinarian is also referred to 
herein as a "treatment effective" amount. 
The active ingredient may be administered by any route appropriate to the 
disorder being treated. Although the compositions of the present invention 
are preferably injected into one or more muscles of the mammal being 
treated, other acceptable methods, e.g., surgical implantation, will 
become apparent to those skilled in the art. It is readily appreciated 
that the preferred route may vary with the disorder being treated. 
While it is possible for the active ingredient to be administered as the 
pure or substantially pure cells, it is preferable to present it as a 
pharmaceutical formulation or preparation. 
The formulations to be used in the practice of the present invention, both 
for veterinary and for human use, comprise myogenic cells, as described 
above, together with one or more pharmaceutically acceptable carriers 
therefor and optionally, other therapeutic ingredients. The carriers must 
be "acceptable" in the sense of being compatible with the other 
ingredients of the formulation and not deleterious to the recipient 
thereof. Such carriers are well known to those skilled in the art of 
pharmacology. Desirably, the formulation should not include other 
substances with which myogenic cells are known to be incompatible. In 
accordance with acceptable pharmacological standards. All methods include 
the step of bringing into association the active ingredient with a carrier 
which may constitute one or more accessory ingredients. 
Formulations suitable for administration by injection conveniently comprise 
sterile aqueous solutions of the myogenic cells, which solutions are 
preferably isotonic with the blood of the recipient. Such formulations may 
be conveniently prepared by following Good Laboratory Practice to produce 
a pharmacologically acceptable sterile aqueous solution. 
The claimed invention will be further understood with reference to the 
following examples which are purely exemplary in nature and are not meant 
to be utilized to limit the scope of the invention. 
EXAMPLES 
1. Animals 
Heterozygous breeders of the hosts were designated C57BL/6J-+/dy.sup.2J 
gpi-1b/lb (Bar Harbor Laboratories, Bar Harbor, ME). Host mice could be 
phenotypically normal (+/+, +/dy.sup.2J), or dystrophic (dy.sup.2J 
/dy.sup.2J), with murine dystrophy being inherited in an autosomal 
recessive pattern. These mice produced glucosephosphate isomerase GPl-lBB 
(mol. Wt. 134,000 daltons), which was used as a genotype marker to 
identify the host cells. Mice of either sex and aged 20 days were used as 
hosts. At this age, dystrophic symptoms began to appear. 
2. Immunosuppression 
In this study, host mice were primed (Rucker et al., Transplantation, 34: 
356, (1982)) one week with CsA injected subcutaneously everyday at 50 
mg/kg body weight before receiving myoblasts. The same CsA treatment 
continued until the sacrifice of the host mice. Since the CsA stock 
solution has a concentration (15 mg/ml) higher than the tolerable level 
for G8 myoblast (25 ug/ml), CsA was injected in small volume (from about 
70 to about 100 ul) on the back of the mice away from the donor cells. 
3. Donor Myoblasts 
G8-1 Cell Line. It is reported by Christian et al., Science 196:995 (1977) 
that: "The clonal line, G-8, was subcultured from M114, an uncloned 
myogenic cell line which arose spontaneously in a culture of cells 
dissociated from Swiss Webster mouse hindlimb muscle. Subculture took 
place after the M114 cells underwent approximately six generations. The 
G-8 cells were subcultured 15 time (an estimated 50 cell divisions) 
without loss of the ability to form myotubes. Multinucleated spherical 
cells also are found in some older cultures. Well-differentiated G-8 
myotubes possess striations and closely resembled normal mouse myotubes in 
morphology. Many G-8 myotubes contract spontaneously. Clonal myotubes were 
similiar to cultured mouse embryomyotubes with respect to acetylcholine 
sensitivity and other membrane resistance characteristics. They differ 
primarily in the resting membrane potential and in the variation in 
sensitivity to acetylcholine at different sites on the membrane surface. 
This may indicate that normal myotubes mature more quickly in vitro than 
do clonal myotubes. The clonal myotubes can form synapses with neurogenic 
cells." 
The G8-1 myoblasts used in the examples, below, are subclones from the G8 
cell line. They can be purchased from The American Type Culture Collection 
(ATCC) at a serial passage of about fourteen. These cells retain the above 
capabilities as studied in our laboratories. Cell doubling time is about 
22.5 hours. When seeded at optimal concentration (2.times.10.sup.6 in 25 
ml of culture medium in a 72 cm.sup.2 Falcon flask), the myoblasts will 
undergo mitosis for about 48 hours and then start to fuse. 
Maintenance of G8-1 myoblasts. The cell culture prodedure used to culture 
the G8 myoblasts is modified from that of Christian et al. supra. The 
cells, purchased in lots of 10.sup.6 from ATCC, are incubated at 
36.degree. C. in 10% CO.sub.2 in G8-1 medium consisting of 95% Dulbecco's 
MEM with 5% fetal bovine serum, penicillin (50 units/ml), sodium salt, and 
streptomycin sulfate (50 ug/ml) in 72 cm.sup.2 Falcon plastic flasks, with 
collagen. The culture medium is changed overnight to remove the 
dimethyl-sulfoxide and dead cell debris. Initial cell concentration was 
2.times.10.sup.6 in 25 ml per 72 cm.sup.2 flask. They are further cultured 
in fresh medium for 48 hours. When cultures become 60% confluent and just 
before cell fusion occurs, the myoblasts are dissocitated with 5 ml of 
0.02% crude trypsin in Hank's balance salt solution (Ca.sup.2+ and 
Mg.sup.2+ - free). Cell dissociation is hastened with occasional shaking 
and gentle scraping with a rubber policeman. Generally, dissociation is 
completed in 5 minutes. The action of trypsin is stopped immediately by 
adding an equal amount of horse serum or fetal calf serum. Myoblasts are 
settled with mild centrifugation and the serum which is antigenic, is 
replaced by Dulbecco's MEM. A cell count is made on a haemocytometer after 
the cells are distributed homogenously in the solution by gentle shaking. 
The cells are then centrifuged at 180 g for 7 minutes. The supernatant is 
discarded and the cells are ready for transplant. 
For regular maintenance, half of the cells are frozen and stored in 10% 
dimethyl-sulfoxide in mouse medium at each "split". About 10.sup.6 cells 
per ml are frozen first in the freezer and then in the Revco deep freezer 
(-90.degree.). 1 ml of cells are stored in 2 ml vials. The remaining half 
of the cells are used to maintain the culture. They are sub-cultured in 
mouse medium at 20-fold lower cell concentration and incubated in 10% 
CO.sub.2. Cell fusion is avoided by subculturing before 60% confluence and 
by not feeding the fusion medium (2% fetal bovine serum, 98% Dulbecco's 
MEM). Yaffe (Research In Muscle Development and the Muscle Spindle, 
Banker, B. et al. p. 110-112, Excepta Medica: N.Y. 1972.) indicates that 
rat cells grown in nutritional medium supplemented with 20% fetal bovine 
serum and 10% embryo extract proliferate but do not fuse until they become 
very crowded (REF). 
4. Myoblast Transplant 
Nine dystrophic (C57BL/6J-dy .sup.2J dy.sup.2J) mice and twelve normal 
littermates received normal myoblasts injections into their hindlimb and 
intercostal muscles. Donor myoblasts were clones of G8 cell line (ATCC) 
originally derived from limb muscles of the Swiss Webster mice (Christian 
et al., Science, 196: 995, (1977)). 
Injection was conducted in a sterile laminar flow hood in a microsurgery 
room equipped with U-V lights. Aseptic precautions were taken. Host mice 
were awake without anesthesia and were restrained during injection. About 
8.times.10.sup.6 donor myoblasts were loaded with mild suction into a 
tuberculin syringe via a 30 gauge needle that was sterile. About 10.sup.6 
myoblasts were injected into each of the following muscle groups on both 
sides of the host: the quadriceps, femoris, hamstrings, adductors, 
extensors, flexors, peroneal and the external intercoastal muscles. The 
needle was slowly withdrawn as the myoblasts were injected. The 
unavoidable minor damages to fine nerve branches, capillaries, and muscle 
fibers would trigger axonal sprouting, capillary reformation and muscle 
regeneration. The wound sealed by itself as the needle was retracted, 
leaving the muscles "filled" with the pre-determined quantity of 
myoblasts. 
5. Monitor of Behavior and Locomotion 
Two to four months later, eleven of the dystrophic mice showed such 
behavioral improvement that their locomotive patterns were 
indistinguishable from those of the unoperated normal mice. This 
improvement is illustrated by use of the photographs in FIG. 2. Sporadic 
flexion and flaccid extension of their hindlimbs were not seen (FIG. 2A). 
They were able to use their hindlimbs and toes (FIG. 2B). Their hindlimb 
muscles were strong enough to support them and to allow them to balance 
themselves on a glass rod (FIG. 2C). Occasionally they would still walk on 
duck feet. The mouse could now run. Muscle bulk was increased in both legs 
and in the chest. Two other mice showed intermediate improvement 
indicating that there were some functional recovery of the muscles. 
However, when they were tested on the glass rod their hindlimbs were not 
strong enough to hold on to the glass rod. Sporadic flexion and flaccid 
extension of the hindlimbs could still be demonstrated. The remaining 
eight dystrophic mice did not show significant behavioral improvement. 
Normal littermates treated similarly were hyperactive, showed enlarged leg 
and intercoastal muscles, but were otherwise normal. 
Whereas the untreated dystrophic mouse dies at about eight months after 
birth, four dystrophic mice treated in accordance with the claimed 
invention, have survived over ten months with significant muscle 
improvement. 
6. Monitor of Muscle Genotypes 
The survival of G8 clonal myoblasts in muscles of CsA-treated or 
non-treated C57BL/6J normal or dystrophic mice was also examined two 
months post-operatively. Host and donor cells exhibited different genotype 
markers, i.e., muscle isoenzeymes of glucosephosphate isomerase (GPI). 
Donor G8 myoblasts produced GPI-1AA and host cells produced GPI-1BB. All 
of the injected muscles of the CsA-treated normal mice showed GPI-1AA, 
indicating the survival of donor myoblasts in these host muscles. 
Similarly, all of the injected muscles of the CsA-treated dystrophic mice 
showed GPI-1AA (FIG. 3). GPI-1AB was also observed in five test muscles, 
indicating that donor myoblasts fused with host satellite cells. (Data not 
shown in FIG. 3.) GPI-1AA was not present in the myoblast-injected muscles 
of the normal or the dystrophic mice without CsA treatment (FIG. 3). Only 
GPI-1BB representing the host cells was observed in the agarose gell 
electrophoresis. Donor myoblasts did not survive without 
immunosuppressant. (b) The survival and development of donor cells in the 
host muscles were also demonstrated in another series of experiments in 
which only the right legs received myoblast injections, with the left legs 
serving as controls. The injected leg showed muscle enlargement (FIG. 4) 
which was not observable in the contralateral leg. Such muscle enlargement 
was present in the CsA-treated hosts but not in those without 
CsA-treatment, regardless of whether the host was normal or dystrophic. 
These results were obtained from twelve mice from each of the four groups 
two months after myoblast injection. 
7. Monitor of Muscle Phenotypes 
FIG. 5 shows cross-sections of the tibialis anterior muscle of both normal 
and dystophic mice with varing CsA treatment. Histologically, there was no 
indication of the presence of donor cells in the myoblast-injected normal 
muscles without CsA treatment (FIG. 5A). These muscle preparations, 
showing polyclonal myofibers with peripheral nuclei and minimal 
intercellular connective tissue, were as normal as any intact normal 
controls. Similarly, the dystrophic muscles receiving myoblast injections 
but no CsA treatment (FIG. 5B) did not differ from the intact dystrophic 
controls. However, both CsA-treated normal (FIG. 5C) and dystrophic (FIG. 
5D) muscles showed immature and developing myogenic cells that were not 
observed in non-treated preparations and were thus likely to be donor in 
origin. Two months was not long enough for all of the donor cells to 
mature, (See for example, B. M. Carlson: In Mauro A (ed): Muscle 
Regeneration, Raven press, p. 57, (1979) and Carlson et al., In Muro (ed): 
Muscle Regeneration, p. 493, Raven Press, New York, (1979)). Nonetheless, 
there was a significant improvement in muscle structure in eleven of the 
CsA-treated dystrophic mice (FIG. 5D) as compared to the non-treated 
dystrophic ones (FIG. 5B), both receiving normal myoblasts. Dystrophic 
characteristics such as muscle fiber splitting, central nucleation, 
phagocytic necrosis, variation in fiber shape and size, and increase in 
intercellular connective tissues were rarely present in the CsA-treated 
dystrophic muscle receiving normal myoblasts. 
The invention has been described in detail, including the preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art may make modifications and improvements upon consideration of the 
specification and drawings as described herein.