FIELD OF THE INVENTION
The present invention is a method for preconditioning healthy donor's myoblasts in vitro before transplantation thereof in compatible patients suffering of recessive myopathies, particularly of muscular dystrophy. This in vitro preconditioning improves the success of the transplantation while not requiring an in vivo preconditioning of the patient's muscle by irradiation or by administering muscular toxin.
BACKGROUND OF THE INVENTION
Duchenne muscular dystrophy (DMD) is a progressive disease characterized by the lack of dystrophin under the sarcolemmal membrane6,19,28,37. One possible way to introduce dystrophin in the muscle fibers of the patients to limit the degeneration is to transplant myoblasts obtained from normal subjects30,34,35. Several groups have tried myoblast transplantations to DMD patients but poor graft success was observed17,22,24,38. Even in experimental myoblast transplantation using mdx mice, an animal model of DMD10,25,29, large amount of dystrophin-positive fibers were observed only when nude mdx mice were previously irradiated to prevent regeneration of the muscle fibers by host myoblasts32,43. High percentage of dystrophin-positive fibers was also observed in mdx mice immunosuppressed with FK 506 and in SCID mice, in both cases muscles were previously damaged by notexin injection and irradiated23,27. These results indicate that to obtain successful myoblast transplantation, it is necessary to have not only an immunodeficient mouse or a mouse adequately immunosuppressed but also a host muscle which has been adequately preconditioned. It is, however, impossible in clinical studies to use damaging treatments such as marcaine, notexin and irradiation. If good myoblast transplantation results can be obtained without using such techniques, this would be very helpful for myoblast transplantation in humans.
Recently there has been an increasing interest on the effects of basic fibroblast growth factor (bFGF) and other growth factors on myoblast cultures and myoblast cell lines1,4,5. Basic FGF has been reported to both stimulate proliferation and inhibit differentiation of skeletal myoblasts in vitro15,16. Other growth or trophic factors like insulin growth factor I, transferrin, platelet-derived growth factor, epidermal growth factor, adrenocorticotrophin and macrophage colony-stimulating factor as well as C kinase proteins activators or agonists by which the effect of bFGF is mediated20 may also have similar or even better effects than bFGF on the success of myoblast transplantation7. The use of these stimulating properties to enhance the success of transplantation by in vitro preconditioning of donor's cells and to replace at least partially the use of previously known methods of in vivo preconditioning of recipients' cells has never been suggested before.
Furthermore, it has been recently published by Overall and Sodek (1996) that concanavalin A increased the secretion of metalloproteases by fibroblasts. Since these enzymes are believed to be present in primary myoblasts cultures, and since they may be responsible for the degradation of the extracellular matrix, it would be desirable to precondition the myoblasts in the presence of both a growth factor and an inducer of the production of metalloproteases, to increase the distance of migration of the transplanted myoblasts and to increase the number of fused myoblasts expressing muscle functional proteins. An attractive alternative would be to use donor myoblasts wherein a gene expressing a metalloprotease is inserted.
Metalloproteases are enzymes necessary for tumor invasion, for cell migration45, and for restructuration of extracellular matrix during normal tissue remodelization46. Matrilysine and gelatinase A are metalloproteases involved in tissue invasion of a plurality of cancer types47. The presence of gelatinase A in its active form has been correlated with the generation of new muscle fibers, during muscle degeneration-regeneration process48. It has been shown that the activity of gelatinase A can induce cell migration by cleaving laminin-5, an extracellular matrix component, thereby exposing a pro-migratory kryptic site49.
From the foregoing, it is really apparent that a compound capable of stimulating the expression of a metalloproteases involved in an extra-cellular restructuration, such as phorbol ester or concanavalin A, would be useful to increase the success of transplantation of myoblasts. Since metalloproteases appear to be secreted in the culture medium, it would also be useful to test if metalloproteases such as matrilysine, gelatinase A, or other metalloproteases of the same class, could be injected directly with myoblasts in recipient muscle for the same purpose.
STATEMENT OF THE INVENTION
The present invention relates to a method of in vitro preconditioning of myoblasts harvested from healthy donor's biopsy prior to their transplantation in patients affected by recessive myopathies, particularly by Duchenne muscular dystrophy (DMD). In a DMD animal model (mdx), compatible donor mouse myoblasts were grown in culture with muscular growth or trophic factors, particularly, basic Fibroblast Growth Factor (bFGF), before transplanting them in muscles of mdx mice without any previous damaging treatment. A four fold increase in the percentage of muscle fibers expressing dystrophin, which is indicative of functional muscle cells, was obtained with pretreatment with bFGF. These experimental results are expected to verify in naturally occurring dystrophy or other types of recessive myopathies in animal and human subjects, since the mdx mouse is an animal model wherein muscular dystrophy is naturally occurring.
Furthermore, culturing the myoblasts in the presence of concanavalin A during two to four days prior to transplantation increases by 3 to 4 fold the distance of migration of the transplanted cells into the recipient tissue. Another inducer of the expression of metalloproteases, phorbol ester has been also used and reproduced the same result as for concanavalin A (increase migration and increased number of fused cells expressing a reporter gene). Recombinant myoblasts expressing metalloproteases also produced the same result.
It is therefore an object of the invention to provide a method wherein cultured myoblasts are transplanted in the presence of a metalloprotease. The production of metalloproteases may be induced during the period of culturing of primary myoblast cultures with or without the preconditioning step in the presence of muscle growth factor. Alternatively, the metalloproteases may be expressed by recombinant myoblasts or injected concurrently with the transplanted myoblasts. Transplantation of cells along with a matrix degrading amount of metalloproteases and transplantation of recombinant cells expressing these enzymes are not limited to myoblasts, but could rather be adapted to any type of transplantated cells.
In accordance with the present invention is provided a method of increasing the number of transplanting donor's myoblasts which are capable of fusing with the myoblasts of a recipient individual suffering of a myopathies, which comprises the steps of: growing said donor's myoblasts in an appropriate culture medium in the presence of fibroblasts and of an agent inducing an increased secretion of an enzyme involved in extracellular matrix destruction prior to injecting said medium, donor's myoblasts and fibroblasts to said recipient individual.
Alternatively is provided a method, wherein the donor's myoblasts are recombinant myoblasts expressing a gene coding for said enzyme.
Is further provided a method which comprises reproducing one of the above methods, and combining to the inducer agent a growth or trophic factor to increase the multiplication of said healthy myoblasts.
In a specific embodiment, said myopathy is Duchenne muscular dystrophy.
In a preferred embodiment, donor's myoblasts consist of a primary myoblast culture obtained from culturing of an enzymatic cell dispersion of donor's muscle biopsy.
It has been observed that growing of primary cultures of donor's myoblasts, which contain fibroblasts, in the presence of a growth or trophic factor is an in vitro preconditioning step that replaces at least in part an in vivo preconditioning of said recipient individual's muscular tissue by irradiation or by administering a muscular toxin.
The growth or trophic factor is selected from the group consisting of basic fibroblast growth factor (bFGF), insulin growth factor I, transferrin, platelet-derived growth factor, epidermal growth factor, adrenocorticotrophin, macrophage colony-stimulating factor, protein kinase C activators, agonists thereof, and combinations thereof.
In a preferred embodiment, the growth or trophic factor is basic fibroblast growth factor (bFGF)
In a more preferred embodiment, the primary myoblast culture is grown in the presence of 100 ng of recombinant human bFGF per milliliter of culture medium for a period of time of about 48 hours before transplantation, whereby a four fold increase of the number of functional muscular cells is obtained.
In still a preferred embodiment, the enzyme involved in the extracellular matrix destruction is a metalloprotease such as matrilysine and gelatinase A and the inducer agent is phorbol ester or concanavalin A.
In a most preferred embodiment, the inducing agent is concanavalin A (Con A).
In a specific embodiment, growing primary myoblast cultures in the presence of 20 μg/ml of Con A for 48 hours resulted in a 3-4 fold increase of the migration distance of transplanted myoblasts and of fused myoblasts.
In the most preferred embodiment, primary myoblast cultures are cultured for two days in the presence of both Con A and bFGF, which would result in a superior transplanting success when compared to each treatment alone.
DESCRIPTION OF THE INVENTION
Although the present trend on research for the treatment of DMD seems to be towards gene therapy, rather than cell therapy, there is still a great deal of work to be done in animal models before either approach, or a mixture of both approaches will be required for the treatment of inherited myopathies such as DMD32,34.
No satisfactory level of dystrophin expression was obtained following myoblast transplantation not only in clinical trials but also in animal experiments not using irradiation33 combined with marcaine or notexin destruction of the muscle26,27. These techniques are, however, too damaging, too invasive or too risky to be used in clinical trials. Basic FGF has been reported to both stimulate proliferation and inhibit differentiation of skeletal myoblasts by suppressing muscle regulatory factors such as MyoD and myogenin12,41. Expression of bFGF has been examined in regenerating skeletal muscles by immunohistochemistry and in situ hybridization, and found to be up-regulated compared to non-injured muscles3,11. Increased skeletal muscle mitogens have also been observed in homogenates of regenerating muscles of mdx mice3. There are increased levels of bFGF in extracellular matrix of mdx skeletal muscles13, mdx satellite cells associated with repair3 and such cells respond more sensitively to exogenous addition of bFGF14. There is a high degree of homology between bFGF from various species2 therefore recombinant human bFGF is active on mouse cells9. In the present series of experiments, myoblasts were pretreated with recombinant human bFGF to increase their proliferation and to verify whether such treatment which is less invasive could have beneficial effects on myoblast transplantation.
In our experiments, primary myoblast cultures from the same donors were grown with or without bFGF and transplanted simultaneously to both tibialis anterior (TA) muscles of the same mice. This seems to be a good model to verify the effect of bFGF because the same primary myoblast cultures, the same grafting conditions and the same immunosuppressive state were used. Comparing both TA muscles, in all treated mdx mice, the percentage of β-galactosidase-positive fibers (this enzyme being a reporter gene) were significantly higher in left TA muscles cultures (with bFGF) than in right TA muscles cultures (without bFGF). In the muscles grafted with myoblasts grown with bFGF, the average percentage of hybrid fibers was 34.4%, with two muscles containing over 40% of donor or hybrid fibers. These are the best results ever reported following myoblast transplantation without notexin or irradiation treatment.
In the present study, myoblasts were incubated with bFGF during 48 hours and about 5 millions of these cells (about 1.75 million myogenic cells) were injected in one TA muscle. The same number of myoblasts not incubated with bFGF was injected in the control contralateral TA muscle. The higher percentage of β-galactosidase/dystrophin-positive fibers was therefore not the consequence of a higher proliferation of the myoblasts in vitro before the transplantations.
Our in vitro results indicate that an incubation during 2 days with bFGF did not significantly modify the total number of cells and the percentage of myogenic nuclei. Basic FGF did, however, significantly inhibit the fusion of myoblasts in vitro. This resulted in a small but significant increase (35%) of the percentage of myoblasts among mononuclear cells. This increase seems too small to account alone for the more than four fold increase of effectiveness of myoblast transplantation produced by bFGF. Recently both Partridge7 and Karpati's24 group reported that a high percentage (up to 99% in Partridge's results) of the myoblasts injected in a mouse die within 5 days. This dramatic result does not seem attributable to immunological problems since it was observed following autotransplantation24 or transplantation in nude mice7. In our experiments, although there were slightly more cells surviving three days post-transplantation for the cultures treated with bFGF, the difference did not reach a significant level and does not seem to account alone for the 4 fold beneficial effect observed 30 days post transplantation.
Basic FGF is thought to regulate myogenesis during muscle development and regeneration in vivo3. The increase percentage of muscle fibers containing the donor gene produced by the addition of bFGF may seem surprising since bFGF was reported to inhibit differentiation of myoblasts in vitro1,13. Basic FGF is, however, one of many growth factors which are liberated following muscle damage7. These factors, all together, certainly increase myoblast proliferation and eventually muscle repairs. We have also observed that following a two day incubation with bFGF of primary myoblast cultures, myoblast fusion occurred within a few days after removal of bFGF (data not shown). The inhibition by bFGF on myoblast fusion is therefore not irreversible. Basic FGF is already at an increased level in mdx muscle, therefore it is not surprising that direct intramuscular injection did not increase the fusion of the donor myoblasts with the host fibers. In fact, bFGF injected directly in the muscle probably stimulates the proliferation of the host as well as the donor myoblasts and therefore do not favour the donor myoblasts. On the contrary, preliminary stimulation by bFGF of the donor myoblasts in culture may favour these myoblasts to proliferate more and eventually participate more to muscle regeneration than the host myoblasts. Though bFGF stimulates the fibroblasts, which an inconvenience for primary myoblast cultures, incubation of myoblast primary culture during only 48 hours with bFGF did not adversely affect our transplantation results and did on the contrary improve them. If primary myoblast cultures were made fibroblast-free by sub-cloning, it is envisageable to precondition the donors' myoblasts for a longer time and increasing this way the number of cells to be transplanted from a relatively small biopsy.
Although the results obtained following transplantation of myoblasts grown with bFGF are not as good than those obtained using irradiation and notexin27, these results are nevertheless important because no technique to destroy the muscles was used. The proposed in vitro preconditioning method might therefore be used in complete replacement of such in vivo damaging pretreatment of recipient cells, or at least in partial replacement thereof, which will result in a substantial diminution of undesirable effects. The effects of many growth factors and trophic factors on myoblast culture have been reported, it is possible that other factors such as insulin growth factor I, transferrin, platelet-derived growth factor, epidermal growth factor, adrenocorticotrophin and macrophage colony-stimulating factor may also have similar or even better effects than bFGF on the success of myoblast transplantation7. Furthermore, since the effect of bFGF is mediated by proteins kinase C, pharmacological agents used to enhance the activity of these enzymes (like phorbol esters) or mimicking the effect thereof (agonists) might also be used for preconditioning myoblasts. Therefore, at least one of these factors can be used alone or in combination with or without bFGF to enhance the success of myoblast transplantation. While the mechanism involved remains speculative, bFGF seems to improve the long term viability, multiplication and fusion of myoblasts. Our results suggest that pretreatment of myoblasts with bFGF may be one procedure that may increase the success of myoblast transplantation in DMD patients.
The present invention will be further described by way of the following Examples and Figures, which purpose is to illustrate this invention rather than to limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows cross sections of TA muscle of mdx mice 28 days after injection of the transgenic myoblasts. Pairs of serial sections from 3 different muscles of three mice are illustrated. Panels a and b illustrate sections of muscles injected with myoblasts grown without bFGF. Panels c to f illustrate sections of muscles injected with myoblasts grown with bFGF. In each pair, one section was stained for β-galactosidase (panels a, c and e). The other section of the pair was immunostained for dystrophin (panels b, d and f). The muscles injected with myoblasts grown in presence of bFGF contained much more β-galactosidase and dystrophin positive fibers than muscles injected with myoblasts grown without bFGF. Most muscle fibers expressing β-galactosidase were dystrophin-positive. In each pair of panels, the same muscle fibers are identified by the same numbers. Scale bar is 100 μm.
FIG. 2 shows the number of muscle fibers positive for X-Gal counted after an injection of 500 000 donor's cells in one site of the tibialis anterior of recipient mice. Imm 7 neo: expresses neomycin. Imm 7 Matrilysine: expresses neomycin and matrilysine. Tn I βgal: untreated transgenic mouse myoblasts expressing β-Gal. Tn I βGal+TPA: transgenic mouse myoblasts/β-Gal treated with phorbol ester.