| Method for in vitro preconditioning of myoblasts before transplantation -> Monitor Keywords |
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Method for in vitro preconditioning of myoblasts before transplantationRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.), Eukaryotic CellMethod for in vitro preconditioning of myoblasts before transplantation description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070178077, Method for in vitro preconditioning of myoblasts before transplantation. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a divisional of U.S. patent application Ser. No. 10/105,815, filed Mar. 21, 2002, which in turn was a continuation of U.S. Patent application Ser. No. 09/284,605, filed Jun. 9, 1999, which in turn was a U.S. national phase under 35 U.S.C. .sctn.371 of PCT/CA97/00774, which was filed on Oct. 17, 1997. Priority under 35 U.S.C. .sctn.120 is claimed to Ser. No. 10/105,815, to Ser. No. 09/284,605, and to PCT/CA97/00774. Priority under 35 U.S.C. .sctn.119(e) is claimed through PCT/CA97/00774 to U.S. provisional application Ser. No. 60/028,692, which was filed on Oct. 18, 1996. The disclosure of each of the foregoing applications is incorporated by reference herein in its entirety. FIELD OF THE INVENTION [0002] 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 [0003] Duchenne muscular dystrophy (DMD) is a progressive disease characterized by the lack of dystrophin under the sarcolemmal membrane.sup.6,19,28,37 . One possible way to introduce dystrophin in the muscle fibers of the patients to limit the degeneration is to transplant myoblasts obtained from normal subjects.sup.30,34,35. Several groups have tried myoblast transplantations to DMD patients but poor graft success was observed.sup.17,22,24,38. Even in experimental myoblast transplantation using mdx mice, an animal model of DMD.sup.10,25,29, 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 myoblasts.sup.32,43. 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 irradiated.sup.23,27. 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. [0004] 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 lines.sup.1,4,5. Basic FGF has been reported to both stimulate proliferation and inhibit differentiation of skeletal myoblasts in vitro.sup.5,6. 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 mediated.sup.20 may also have similar or even better effects than bFGF on the success of myoblast transplantation.sup.7. 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. [0005] 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. [0006] Metalloproteases are enzymes necessary for tumor invasion, for cell migration.sup.45, and for restructuration of extracellular matrix during normal tissue remodelization.sup.46. Matrilysine and gelatinase A are metalloproteases involved in tissue invasion of a plurality of cancer types.sup.47. The presence of gelatinase A in its active form has been correlated with the generation of new muscle fibers, during muscle degeneration-regeneration process.sup.48. 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 site [0007] 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. SUMMARY OF THE INVENTION [0008] 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. [0009] 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. [0010] 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. [0011] 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. [0012] Alternatively is provided a method, wherein the donor's myoblasts are recombinant myoblasts expressing a gene coding for said enzyme. [0013] 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. [0014] In a specific embodiment, said myopathy is Duchenne muscular dystrophy. [0015] 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. [0016] 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. [0017] 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. [0018] In a preferred embodiment, the growth or trophic factor is basic fibroblast growth factor (bFGF). [0019] 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. [0020] 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. [0021] In a most preferred embodiment, the inducing agent is concanavalin A (Con A) Continue reading about Method for in vitro preconditioning of myoblasts before transplantation... Full patent description for Method for in vitro preconditioning of myoblasts before transplantation Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for in vitro preconditioning of myoblasts before transplantation patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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