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 μg/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.

What is claimed is:  
     
         1 . A method for increasing the number of transplanted functional donor's myoblasts which are fused with non-functional myoblasts of a recipient individual suffering of a myopathy, which comprises the step of growing in vitro said donor's myoblasts in a appropriate culture medium in the presence of fibroblasts and of an agent inducing the secretion of an enzyme involved in extracellular matrix destruction, prior to injecting a mixture comprising said donor's myoblasts and induced enzyme into said recipient individual's muscle, whereby a functional muscle is at least in part restored.  
     
     
         2 . A method for increasing the number of transplanted functional donor's myoblasts which are fused with non-functional myoblasts of a recipient individual suffering of a myopathy, which comprises the steps of: inserting into said donor's myoblasts a gene construct capable of expressing an enzyme involved in extracellular matrix destruction, obtaining thereby recombinant donor's myoblasts, and growing said recombinant donor's myoblasts in an appropriate culture medium, prior to injecting said recombinant donor's myoblasts into said recipient individual's muscle, whereby a functional muscle is at least in part restored.  
     
     
         3 . A method of  claim 1 , wherein said enzyme is a metalloprotease.  
     
     
         4 . A method of  claim 2 , wherein said enzyme is a metalloprotease.  
     
     
         5 . A method of  claim 3 , wherein said metalloprotease is Gelatinase A or Matrilysine.  
     
     
         6 . A method of  claim 4 , wherein said metalloprotease is Gelatinase A or Matrilysine.  
     
     
         7 . A method of  claim 1 ,  3  or  5 , wherein said agent is Concanavalin A or phorbol ester.  
     
     
         8 . A method as defined in any one of  claims 1  to  7 , wherein said culture medium further comprises a growth or trophic factor for increasing the multiplication of said donor's myoblasts.  
     
     
         9 . A method as defined in  claim 8 , wherein said 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.  
     
     
         10 . A method as defined in  claim 9 , wherein said factor is bFGF.  
     
     
         11 . A method as defined in any one of claims  1 ,  3 ,  5 ,  7 ,  8 ,  9 , and  10 , wherein said donor's myoblasts are obtained for a primary myoblast culture resulting from culturing a cell dispersion of donor's muscle biopsy.  
     
     
         12 . A method as defined in  claim 11 , wherein said primary myoblast culture is grown in the presence of 100 ng of recombinant human basic fibroblast growth factor per milliliter of culture medium for a period of time of about 48 hours before transplantation.  
     
     
         13 . A method as defined in  claim 11 , wherein said primary myoblast culture is grown in the presence of 100 ng of recombinant human basic fibroblast growth factor and 20 μg Concanavalin A per milliter of culture medium for a period of time of about 48 hours before transplantation.  
     
     
         14 . A method as defined in any one of  claims 1  to  13 , wherein said myopathy is Duchenne muscular dystrophy.  
     
     
         15 . A method for increasing the number of transplanted functional donor's cells which are fused with corresponding non-functional cells of a recipient individual's tissue, which comprises the steps of: inserting into said donor's cells a gene construct capable of expressing an enzyme involved in extracellular matrix destruction, obtaining thereby recombinant donor's cells, and growing said recombinant donor's cells in an appropriate culture medium, prior to injecting said recombinant donor's cells into said recipient individual's tissue, whereby a functional tissue is at least in part restored.  
     
     
         16 . A method of  claim 15 , wherein said enzyme is a metalloprotease.  
     
     
         17 . A method of  claim 16 , wherein said metalloprotease is Matrilysine or Gelatinase A.