Method for in vitro preconditioning of myoblasts before transplantation

Abstract

A method of pretreating healthy donor's myoblast cultures with growth or trophic factors like basic fibroblast growth factor (bFGF) and with concanavalin A on transplantation to subjects suffering of myopathy like muscular dystrophy is disclosed and claimed. Recipient muscles show a higher percentage of functional cells, a four-fold increase, demonstrated by the higher incidence of dystrophin-positive fibers, and does not require previous preconditioning of recipient muscles by irradiation or toxin administration. The recipient subjects were immunosuppressed with FK 506. When growing myoblasts with 20 mug / ml concanavalin A or 100 ng / ml TPA for two to four days, migration of donor cells in recipient tissue was increased by 3-4 fold. This suggests that, when using primary cultures, metalloproteases are secreted by fibroblasts, resulting in a greater degradation of the extracellular matrix. Both metalloproteases and bFGF appear beneficial for the success of the transplantation. The use of recombinant myoblast expressing metalloproteases is also contemplated.

Description

[0001] 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.[0002] 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-positi...

AI Technical Summary

Benefits of technology

[0010] 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.

[0021] In a specific embodiment, growing primary myoblast cultures in the presence of 20 .mu.g / 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.

Problems solved by technology

It is, however, impossible in clinical studies to use damaging treatments such as marcaine, notexin and irradiation.

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