WO1993024151A9

WO1993024151A9 - Arterial introduction of myoblasts

Info

Publication number: WO1993024151A9
Application number: PCT/US1993/004812
Authority: WO
Filing date: 1993-05-20
Publication date: 1996-08-15

Abstract

A method for treating an abnormal muscle of a mammal which involves introducing myoblasts into an artery of the mammal.

Description

Arterial Introduction of Myoblasts

Background of the Invention Myoblasts are immature muscle cells. They can be cultured in vitro and cloned. In vivo, skeletal myoblasts with single nuclei (mononucleate) fuse to form muscle fibers (myofibers) with multiple nuclei

(multinucleate) . In contrast to skeletal muscle, heart muscle (myocardium) is not multinucleate; its cells are, however, tightly arrayed in a syncytial pattern.

Myoblasts have been observed to migrate across the basal lamina. It has been indicated that they do not migrate from the blood vessels into surrounding muscle. They have been shown to migrate short distances within muscle. Injection of skeletal myoblasts through the skin directly into skeletal muscle of both rodents and humans has resulted in migration of myoblasts and subsequent formation of multinucleate cells combining the nuclei of the injected cell (donor) and the host cell (recipient) . The multinucleate cells have been observed to produce genetically-coded products of both the donor and recipient cells. This has been identified as a possible treatment for muscular dystrophy.

Both skeletal and heart muscle have been shown to be capable of incorporating and expressing genetic material in the form of a plasmid. There are numerous diseases involving skeletal or heart muscle cells. In some diseases, both skeletal muscle and heart muscle are abnormal (as in Duchenne muscular dystrophy) , in other diseases, only the heart muscle is abnormal (as in cardiomyopathies associated with coronary artery blockages, viral illness, and chemotherapy) , while in still other diseases, only the skeletal muscle is abnormal (as in McArdie's disease) . Many of these diseases have limited treatments. The late stage of cardiomyopathy, for instance, may be intractable to pharmacologic therapy. Attempts have been made to augment muscle contraction by surgically transposing a flap of whole skeletal muscle. Heart transplantation is used in extreme cases. Studies have indicated that human and non-human cells can be cultured and administered therapeutically to human patients, either topically or intravascularly, for treatment of such conditions as diabetes, cystic fibrosis, leukemia, and burns.

Summary of the Invention

The invention features a method for treating an abnormal muscle of a mammal, by introducing myoblasts into an artery supplying the abnormal muscle. The abnormal muscle may be skeletal muscle or heart muscle; the myoblasts may be autologous skeletal myoblasts or non-autologous skeletal myoblasts; and the mammal may have muscular dystrophy or a cardiomyopathy.

The method preferably involves catheterizing a peripheral artery (femoral, brachial, or axillary) or aorta, advancing the catheter into an artery supplying an abnormal muscle (target muscle) , and introducing skeletal myoblasts into the artery supplying the target muscle. The myoblasts are able to migrate across the vascular wall into the target muscle parenchyma. The target muscle may be either skeletal or cardiac, and the skeletal myoblasts may be either autologous or non- autologous. Once within the parenchyma, the donor myoblasts are capable of combining with other donor myoblasts as well as with recipient muscle cells (in the case of skeletal muscle) and only with other donor myoblasts (in the case of cardiac muscle) to form contractile myotubes. In each instance, the myotube can serve to augment the contractility of the target muscle. If the introduced skeletal myoblasts are non-autologous, then the method further includes treatment of the recipient with an immunosuppressive regimen to prevent rejection.

The instant invention provides unique and critical advantages over any previous method. First, the intraarterial route offers the possibility of implanting myoblasts in virtually any target muscle, including those inaccessible by direct intramuscular inoculation, such as the diaphragm. Second, whereas intramuscular inoculation can only seed a small area around the needle tract, intraarterial introduction of cells permits a wide area of muscle coverage. Third, intraarterial administration permits delivery of myoblasts to the myocardium where they may form skeletal myotubes capable of augmenting the contraction of impaired heart muscle cells.

In another aspect, the invention features a method for providing a protein to a mammal by providing a myoblast transfected with DNA encoding that protein, and then introducing the transfected myoblast into an artery of the mammal.

In another aspect, the invention features a method for treating an abnormal muscle of a mammal by providing a myoblast transfected with DNA encoding a molecule capable of reversing that abnormality, and then introducing the transfected myoblast into an artery of the mammal.

In another aspect, the invention features a method for treating an abnormal muscle of a mammal by introducing myoblasts intramuscularly into the myocardium. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description

The drawings are first described. Drawings

Fig. 1 is a set of photomicrographs of skeletal muscle after intramuscular (IM) and intraarterial (IA) introduction of myoblasts tagged with the lacZ gene marker. Panel A: hematoxylin and eosin staining 14 days after IM introduction, showing a needle tract of regenerating cells; bar=40μm. Panel B: x-gal staining for the presence of the lacZ gene marker (black) in the section of skeletal muscle adjacent to the section shown in Panel A, at the same magnification, showing the regenerating cells bearing the lacZ gene marker. Panel C: longitudinal section with x-gal staining, 14 days after IA myoblast introduction showing myotubes bearing the lacZ gene marker (black) ; bar=80μm. Panel D: cross section with x-gal staining, 28 days after IA myoblast introduction at the same magnification as Panel C. Panel E: cross section with x-gal staining, 28 days after IA myoblast introduction, at higher magnification; bar=40μm. Fig. 2 is a set of photomicrographs of heart muscle after IA and IM introduction of myoblasts tagged with the lacZ gene marker. Panel A: x-gal staining for the presence of the lacZ marker (black) , 7 days after IA myoblast introduction (ischemic lesion induced) ; arrows indicate cell margin; bar=20 μm. Panel B: x-gal staining, 7 days after IM myoblast introduction (no ischemic lesion induced) ; bar=40μm. Experimental Data Material and Methods

L6 Mvoblasts. Rat-derived L6 myoblasts (Richler et al., 1970, Dev Biol 23: 1-22) were used as donor cells primarily because this transformed muscle line is easily propagated in culture and readily cloned for preparation of large numbers of labelled donor cells. The standard growth medium for L6 myoblasts is Dulbecco's minimal essential medium (DMEM) with 20% fetal calf serum (FCS) , 2% 1-glutamine and penicillin/streptomycin or Ham's F12 containing 10% FCS and 5 ng/ml fibroblast growth factor (FGF) , 10 ng/ml epidermal growth factor and 10 ug/ml insulin. To verify myogenicity, L6 myotube formation is induced in vitro by allowing myoblasts to grow to confluence and switching growth medium to 2% FCS (Yasin et al, 1977, J Neurol Sci 32: 347-360). L6 cells are cloned by the technique of limiting dilution, calculated to give 10, 1 and 0.1 cells/microwell.

Labelling of L6 mvoblasts. A critical requirement for these studies is a mechanism for labelling donor myoblasts to distinguish them from host cells following injection. The MuLV-derived "bag" virus containing both the neomycin resistance and lacZ genes was prepared in psi-2 packaging cells (Cepco, 1988, Neuron l: 345-353; Price et al., 1987, Proc Natl Acad Sci USA 84:156-60) and used to infect clones of L6 myoblasts following the method of Price et al (Price et al., 1987, Proc Natl Acad Sci USA 84:156-60). Infected L6 cells were then incubated in the neomycin analog g418 to select for stable transfectants which were recloned. Clones expressing lacZ were aliquotted and frozen at -70°C. To identify lacZ-positive cells, tissues are immediately frozen in liquid nitrogen cooled isopentane, cut in serial 16 μm sections, and mounted on slides. Sections were fixed in 0.5% glutaraldehyde and then stained in 1 mg/ml 5-bromo-4-chloro-3-indonyl-D-galactoside (x-gal) overnight. Alternate sections were stained with hematoxylin and eosin.

Skeletal muscle lesion. Lewis rats of approximately 200g were used in all experiments. Foci of muscle injury (for production of regenerating myotubes into which the donor myoblasts could incorporate) were generated by chemolysis injecting 0.5% bupivacaine and 15IU hyaluronidase in 0.5 cc amounts on three successive days directly into the left tibialis anterior muscle (TA) (Hall-Craggs, 1974, Exp Neurol 43:349-358). Within 24-36 hours this induces rapid myonecrosis followed over 2 to 4 days by a brisk proliferation of host myoblasts which align to fuse and form new myotubes. By 4-6 weeks, the repair process is virtually complete. At peak injury, electron microscopy reveals focal plasma membrane lysis with preservation of the basal lamina analogous to that observed in Duchenne muscular dystrophy (Orimo et al., 1991, Muscle and Nerve 14:515-520).

As a control for the chemical lesion, the contralateral side was injected with sterile saline in the same volume. This produces some disruption of myofibers but does not provoke the fulminant myonecrosis and regeneration or the associated inflammatory response seen in the chemical lesion.

Myoblast injections into skeletal muscle. L6 myoblasts were introduced into the skeletal muscle using either intramuscular or intraarterial introduction. The former technique was to serve as a control for the latter.

Direct intramuscular introduction. One day after muscle lesions were created in the TA using either the combination of bupivacaine/hyaluronidase or saline, rats were anesthetized with 30 g/kg of pentobarbital. Approximately 10⁶ freshly trypsinized and rinsed lacZ- positive L6 cells in 0.3 cc of saline were injected into the left TA muscle through a 26 gauge needle. The needle was inserted parallel to the tibia and cells were injected along the length of the needle tract as the needle was withdrawn. Intraarterial introduction. As above, one day after creation of muscle lesions, the rats were anesthetized with 30 mg/kg of pentobarbital. The left flank was shaved, cleansed and opened to expose the aorta. Using a 28 gauge needle bent at a 45° angle, 3 x 10⁶ freshly dissociated and rinsed lacZ-positive myoblasts in 0.5 cc normal saline were injected into the aorta distal to the renal arteries. When necessary, cellulose pledgets were applied to stop bleeding after injection.

Ischemic myocardial lesion. In initial cardiac experiments, an ischemic lesion was induced in the myocardium in the expectation that significant disruption of the myocardial syncytium might be necessary to permit survival of myoblasts and myoblast fusion into myotubes. However, subsequent work showed that myoblast survival and fusion could occur without prior syncytial disruption. Since ischemic lesions significantly weakened the rats, the technique was changed in the later experiments, such that myoblasts were introduced into normal hearts.

To produce an ischemic zone in the heart, a sterile surgical thoracotomy was performed on the rats; through this incision, the left anterior descending (LAD) coronary artery was occluded for thirty minutes with a proline suture to produce selective LAD territory ischemia (Force et al., 1989, Am J Physiol 257: H1204- 1210; Evans et al., 1985, Cardiovas Res 19: 132-138). The aorta was catheterized prior to reperfusion, and a coronary angiogram was performed to evaluate the distribution of blood flow. Histological analysis of the lesion after ischemia documented classical changes of myocardial ischemia and infarction (Evans et al., 1985, Cardiovas Res 19:132-138).

Myoblast delivery into the myocardium. L6 myoblasts were introduced into the myocardium via either intramuscular (control) or intraarterial introduction. Direct intramuscular introduction. Ten minutes after reperfusion, a total of 10⁶ lacZ labelled L6 myoblasts in 0.5 cc normal saline were injected directly into the myocardium, in five injections each of 0.1 cc. Intraarterial introduction. Ten minutes after reperfusion, 10⁶ lacZ labelled L6 myoblasts in 0.5 cc normal saline were injected into the ascending aorta just distal to the origin of the LAD. When the latter method was used, blood flow into the coronary arteries during myoblast injection was increased by brief occlusion of the aorta at the level of the arch.

As noted above, ischemic lesions were only induced during early experiments. Subsequently, the method of injecting myoblasts was unchanged, but no ischemic lesion was induced beforehand.

Immunosuppressio . To prevent allograft rejection, recipient rats were immunosuppressed with daily injections of cyclosporine at 10 mg/kg.

Results: Intramuscular injections into skeletal muscle.

Sections of the right TA (in which the pre-myoblast lesion had been induced with saline only) and of the left TA (in which the pre-myoblast lesion had been induced with bupivacaine/hyaluronidase) were studied for the presence of the lacZ gene marker (using x-gal staining) at 7, 14, and 21 days. The right TA (control) showed no evidence of the lacZ marker. In the left TA, muscle cells staining darkly for the lacZ marker were easily seen along the margins of the injection tract at each time interval. At 7 days, staining of the lacZ marker was confined largely to single cells. By 14 and 21 days, lacZ staining was evident within the cytoplasm of differentiated muscle cells (fig. 1) .

Intraarterial injections into skeletal muscle. Sections of left and right TA were studied for the presence of the lacZ marker (using x-gal staining) at 2 hours and 7, 14 and 28 days. At 2 hours, lacZ-positive cells were evident in both left and right TA's. At 7, 14, and 28 days, lacZ-positive cells were evident in both left and right TA's, both as single cells and as small clusters of cells (fig. 1) . The length of dark-staining regions in some myotubes strongly suggested that more than one donor nucleus was incorporated into the same myotube, as illustrated in the longitudinally stained fiber in fig. 1.

In Table 1 are results from the intraarterial introduction of myoblasts into skeletal muscle. At 14 and 28 days, the number of lacZ-positive myofibers was counted in each section of TA. In each section, the number of total myofibers was estimated using an average fiber size (determined from measuring 100 fibers) and the area of muscle analyzed. At 28 days post-myoblast injection, the left TA (in which the bupivacaine/hyaluronidase lesion had been induced) demonstrated, on average, a total of 17 lac-Z positive myofibers per 10⁴ fibers (2562/15 x 10⁶) . In the right TA (which had been injected with saline only) , there were 12 lacZ-positive fibers per 10⁴ fibers examined. The difference in the number of lac-Z positive fibers between the left and right TA's was not statistically significant.

Following the intra-arterial injection, the rats were examined for the presence of L6 myoblasts outside muscle tissue, i.e. in liver, spleen, lung, aortic injection site, or retroperitoneal space. In all rats, the liver and spleen were free of myoblasts. The lung of one of twelve rats showed a small number of lacZ- positive cells. In all rats, the aortic injection site was free of myoblast thrombi. Although there was evidence in some animals of lacZ-positive cells in the retroperitoneal space, this finding could be explained by leakage of cells from the aorta into the retroperitoneum following withdrawal of the injection needle.

The timing of the myoblast migration from the arterial circulation to foci of muscle injury was relatively rapid. Isolated myoblasts were detected in the injured muscle in the as little as two hours after intra-arterial myoblast injection. However, the fact that at 2 hours, clusters of myoblasts could still be detected in arterioles (data not shown) , suggests that not all myoblasts were able to diffuse through the capillary wall to the muscle in two hours or less.

Most lacZ-positive myoblasts were seen in foci of injury; however, single myoblasts were also visualized in uninjured areas of muscle.

Intramuscular injection into myocardium. At one week after the injection of myoblasts into the myocardium, the muscle demonstrated evidence of lacZ- positive cells. The presence in some cases of the lacZ marker along the length of a cell suggested that the myoblasts had fused to form myotubes within the myocardial injury site (fig. 2) . Fusion did not appear to require previous myocardial injury.

Intraarterial injection into myocardium. Five days after intraarterial injection of myoblasts, lacZ- positive cells were detected, singly and in clusters. Further, the presence of the lacZ marker along the length of a cell suggested fusion of myoblasts into myotubes (fig. 2) . Fusion again did not appear to require previous myocardial injury.

Treatment of human subjects

Selection of donors. If autologous myoblasts are to be used, as in the treatment of cardiomyopathy, then the patient may supply his/her own skeletal muscle for the purpose of myoblast culture. If not, then donor- recipient histocompatibility is determined by the following criteria:

1) Donors are histocompatible relatives without significant medical illness. 2) Standard serological HLA type matching for Class I and II major histocompatibility antigens is performed following protocols used for kidney and liver transplantation (Terasaki and McClelland, 1969, Nature 204:998-1000; Vartdal et al., Tissue Antigens 28:301- 312).

3) Patterns of reactivity of mixed, donor-recipient lymphocyte co-cultures in vitro are studied in order to optimize compatibility.

4) Donors are not acceptable if they have serologic evidence of prior infection with selected transmissible viruses (e.g. cytomegalovirus, Ebstein-Barr virus, hepatitis A, B, or C, human immunodeficiency virus, human T-cell leukemia virus) . 5) Donors are not acceptable if they have had an infection within the previous two weeks.

Procedure for donor muscle harvesting. Donor muscle biopsies may be performed in outpatient surgical centers. The usual biopsy site is the quadriceps muscle; other muscles may be selected depending on donor preference and extenuating circumstances. Biopsy is performed in the standard manner known to those skilled in the art, under sterile conditions, using local anesthetics. Muscles samples freshly biopsied for culture are placed immediately into cooled Hank's solution with 10 mN glucose and non-beta lactam antibiotics and transported to the laboratory for culturing. Culture of myoblasts. Myoblast cultures are prepared according to the standard methods known to those skilled in the art. The muscle is trimmed free of connective tissue, weighed, minced, and subjected to three cycles of enzymatic digestion with trypsin and collagenase. The resulting muscle derived cells are then plated to be grown in Dulbecco's minimal essential medium with 20% fetal calf serum (FCS) , pre-selected for ability to grow human myoblasts. Serum is screened in advance for mycoplasma and pathogenic viruses. Geographic source of serum is also ascertained, and attempts are made to purchase serum derived only from fetal calves in regions free of endemic bovine spongiform encephalopathy. The myoblast culture medium is supplemented with 1% 1- glutamine and antibiotics (excluding beta-lactam antibiotics) . After a growth period of about five days, the initial passage cells are harvested and cloned by limiting dilution in 96 well plates (Johnstone et al., 1982, Immunology in Practice, Blackwell Scientific Publications, Oxford, 37-39) . Individual clones are scored over subsequent days for the early appearance of myotubes. When myotubes are identified, the corresponding wells are selected for expansion as clonal myoblasts. In some cases, depending on the amount of donor material initially received, a portion of the muscle is kept under sterile conditions at 4° C in Hank's solution and cultured the following day. In other cases, small pieces of the muscle are frozen in the growth medium described above combined with 10% glycerol, for future thawing and culturing according to this method. Preparation of donor myoblasts for intraarterial injection. The myoblasts are propagated in a sub- confluent state. Approximately three weeks prior to intraarterial injection of myoblasts, the myoblasts are screened for bacterial or mycoplasma infection. Five days before the myoblasts are harvested, antibiotics are omitted from the growth medium. At three days prior to harvesting, the cells are switched to serum-free growth medium prepared according to the methods of Ham et al. (Ham et al., 1989, UCLA Symposia on Molecular and Cellular Biology 93:905-914). This medium is changed twice at 24 hour intervals. The cells are harvested by light trypsinization in the usual manner known to those skilled in the art, 24 hours after the second change of the serum-free medium. The myoblasts are then trypsinized, suspended, and washed twice in saline for further removal of residual calf serum. They are then resuspended in an appropriate concentration of saline (approximately 3-5 x 10⁶ per 500 μl) .

Three or four days before harvesting, an aliquot of the myoblasts is selectively retyped for one or a few major histocompatibility antigens to verify that the MHC type corresponds exactly to that previously determined from the donor lymphocytes. This step verifies that the myoblasts originate from the intended donor. As myoblasts are prepared for harvesting, they are tested at intervals for the presence of the surface NCAM epitope recognized by the monoclonal antibody 5.1H11 (Walsh and Ritter, Nature 2389:60-64). 5.1H11 staining is characteristic of human myoblasts but not fibroblasts and thus can be taken as an index of the myogenicity of cultured cells. Myogenicity is also ascertained by plating a small number of cells at confluent density into a P35 (35 mm) petri dish and counting at seven days both the fusion index (the number of nuclei in myotubes divided by the total number of nuclei) and the total number of myotubes.

Tissue culture facility. All human muscle cultures used in this method are carried out in a facility dedicated exclusively to this purpose.

Equipment includes six-foot laminar flow culture hoods (Nuaire, hepa-filtered) , Forma upright hepa-filtered C0₂ incubators, a refrigerated centrifuge (Sorval RT6000B) , and a shaking water bath (Precision) . The facility is maintained at positive pressure via an overhead pump with a terminal hepa-filter; room air undergoes 80% recirculation through the hepa-filtration system. Only selected laboratory personnel have access to the human muscle tissue culture facility. Testing of donor and recipient sera and cultured myocytes. Sera of potential donors are tested for the following: complete blood count, sedimentation rate, glucose, creatine kinase, total and direct bilirubin, SGOT, alkaline phosphatase, and lactate dehydrogenase. Titers to the following viral agents are determined: hepatitis A, B, and C, human immunodeficiency virus (HIV) , cytomegalovirus (CMV) , and Epstein-Barr virus (EBV) . Recipient sera is screened for CMV, tuberculosis, and syphilis. Approximately three weeks prior to harvesting for donation, a pellet of approximately 10⁵ myoblasts is studied for ultrastructural evidence of the presence of viral particles. Approximately one week prior to harvesting, donor myoblasts are screened for bacteria and mycoplasma.

Intraarterial introduction of mvoblasts. At the time of the myoblast introduction procedure, a 24-hour collection of the recipient's urine is made for urinalysis, total protein, creatinine, and creatine, and a complete blood count is performed. Immediately prior to the procedure, 10 cc of recipient's serum is obtained for storage to permit future antibody studies.

A flexible catheter is inserted into a peripheral artery (preferably femoral; alternatives are brachial artery, axillary artery, or translumbar aorta) of the recipient, using standard techniques well known to those skilled in the art of angiography. The catheter is advanced to the artery supplying the target muscle using standard angiographic techniques. Once the catheter tip is in the desired position, the end external to the recipient is attached to a reservoir containing donor myoblasts in suspension, preferably in saline. Myoblasts may be kept in suspension by a number of different techniques; preferred ones are spin bars in the reservoir or a low concentration of enzymes (e.g. trypsin) in the suspension material to prevent donor myoblast clumping.

The donor myoblasts are delivered to the recipient either by bolus injection, each bolus containing between 20 million and 100 million cells, or by slow arterial infusion, the infusate containing over 100 million cells. The delivery may be done into smaller branch arteries supplying the target muscle, into a larger artery supplying the target muscle, or into the aorta. Note is made that introduction of myoblasts need not be done selectively; if a large number of muscles are to be treated, then injection of myoblasts into the aorta may be done.

Once the donor myoblasts are delivered, the catheter is removed and hemostasis is applied as needed to the artery.

Immunosuppression. If the donor myoblasts are not autologous, then the recipient is given immunosuppression therapy, according to recommended procedures for organ transplantation. Recipients receive cyclosporine 15 mg/kg, 12-24 hours prior to the procedure, and then daily for one week. The dose is then tapered by 5% per week to a maintenance level of 5-10 g/kg/day, given in two equal doses at approximately eight and one o'clock. Cyclosporine blood levels are obtained at day 3 or 4, and then weekly while the recipient is maintained on the drug. Trough levels for whole blood are maintained at 100-450 ng/ml. Blood is monitored weekly for creatinine levels. Liver function tests are done monthly, and include SGOT, SGPT, bilirubin (direct and indirect) and alkaline phosphatase. Complete blood counts are obtained at the time of creatinine level determinations. Immunosuppression is continued as long as it is tolerated by the recipient. Clinical indications. The invention can be used to treat a variety of conditions associated with abnormal muscles in humans. For example, both the skeletal muscle and myocardial manifestations of Duchenne muscular dystrophy, a progressive degenerative disorder of muscle (which has been shown to respond to intramuscular injection of myoblasts) may improve following intraarterial introduction of myoblasts into target muscles. The genetic material of these myoblasts produces a protein not expressed by the patient's own missing or defective gene. Another example is congestive heart failure, which afflicts about 400,000 individuals in the United States (Cupples and D'Agostino, 1987, The Framingham Study: an Epidemiological Investigation of Cardiovascular Disease, eds. Dannel et al., NIH publication no. 87-2703).

Congestive heart failure is caused by generalized damage of the heart muscle, caused by viral infection, chemotherapy, coronary artery blockages, or unknown causes. Since there are few pharmacologic treatments in advanced congestive heart failure, intraarterial introduction of myoblasts into abnormal myocardium, which results in the formation of new myotubes, may improve heart contractility significantly

The invention may also be used to treat autosomal dominant muscle diseases (such as myotonic dystrophy) by introducing into an artery a myoblast transfected with DNA encoded to produce an RNA molecule complementary to the RNA molecule produced by the abnormal muscle.

The invention may also be used for the production in patients of desired proteins (e.g. growth hormone) , by introducing into an artery a myoblast transfected with DNA encoded to produce such a protein.

Table 1. Quantification of Arterially Injected LacZ Positive Muscle Cells

Experiment Normal Saline Chemolvsis Days A B A B

1 14 10 11.20 243 7.67

2 14 43 7.45 127 8.43

3 14 21 6.80 76 10.44

4 14 36 8.12 1 8.13 5 14 0 5.23 197 10.23

6 14 760 18.92 319 10.23

Total 870 57.75 837 40.68

1 28 167 1.82 76 3.81

2 28 1035 3.66 2475 7.18 3 28 32 4.40 11 4.04

Total 2 1236 9.88 2562 15.03

A: Number of lacZ positive myofibers in sections B: Estimated total number of myofibers x 10^"b in sections counted

Table 1. At 14 days, the normalized fraction of LacZ positive muscle cells per total myofibers were 1.5/10,000 (870/57.75xl0⁵) on the saline injected side and 2.1/10,000 (837/40.68xl0⁵) on the chemolysed side. At 28 days these fractions were: 13/10,000

(1236/9.88x10^s) on the saline injected side and 17/10,000 (2562/15.08xl0⁵) on the chemolysed side. LacZ positive cells are not found in uninjected rats.

Claims

We claim:

1. Use of myoblasts in the preparation of a medicament for treatment of an abnormal muscle, preferably skeletal muscle or myocardium, of man or of another mammal by introducing said myoblasts into an artery supplying said abnormal muscle.

2. Use of myoblasts in the preparation of a medicament for the treatment of muscular dystrophy or of cardiomyopathy.

3. Use of autologous skeletal myoblasts for the purpose specified in claims 1 or 2.

4. Use of non-autologous skeletal myoblasts for the purpose specified in claims 1 or 2.

5. Use of myoblasts encoding a molecule capable of reversing said abnormality for the purpose specified in claim 1.

6. Use as specified in claim 5, wherein said molecule is a protein.

7. Use as specified in claim 5, wherein said molecule is RNA.

8. Use of a myoblast transfected with DNA encoding a protein in the preparation of a medicament for providing said protein to a mammal, preferably man, by introduction of said myoblast into an artery of said mammal.

9. A kit for treatment of an abnormal muscle of a human or other mammalian patient, the kit being characterized in comprising: a medicament comprising myoblasts, and at least one flexible catheter adapted for introducing the said medicament into an artery supplying said muscle via femoral artery, auxiliary artery, brachial artery, or lumbar aorta, through which said flexible catheter is adapted to be passed.

10. A kit according to claim 9, characterized in further comprising fluoroscopy apparatus and a radiopaque intravascular contrast material for directing said flexible catheter to the appropriate artery.