HIGH-EFFICIENCY MYOGENIC CONVERSION OF HUMAN FIBROBLASTS BY ADENOVIRAL VECTOR-MEDIATED MYOD GENE-TRANSFER - AN ALTERNATIVE STRATEGY FOR EX-VIVO GENE-THERAPY OF PRIMARY MYOPATHIES

Ex vivo gene therapy of primary myopathies. based on autologous transp lantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expande d, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Prim ary human and murine fibroblasts from skin, muscle, or bone marrow wer e infected by an E1-deleted adenoviral vector carrying a retroviral lo ng terminal repeat-promoted MyoD cDNA, Expression of MyoD caused irrev ersible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunode ficiency/beige) mice, where they gave rise to P-galactosidase positive , centrally nucleated fibers expressing human myosin heavy chains. Fib ers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced sa tellite cells. MyoD-converted murine fibroblasts participated to muscl e regeneration also in immunocompetent syngeneic mice. Although antibo dies from these mice bound to adenoviral infected cells in vitro, no i nflammatory infiltrate was present in the graft site throughout the 3- wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.