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.

Abstract

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, 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. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible 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 immunodeficiency/beige) mice, where they gave rise to ␤ -galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ -transduced satellite cells. MyoD -converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory 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. ( J. Clin. Invest.

Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle

“…Rapid loss of dFbs is consistent with other studies of fibroblasts and myoblasts after transplantation into muscle [12,35,36]. The more rapid loss of transplanted dFbs in the NT group compared with myogenically converting dFbs may be due to differential tolerance to transplantation stress, a feature also noted among different myogenic cell populations [37], or a survival advantage associated with activation of a myogenic program.…”

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

“…To confirm these results, we analyzed primary myoblast cell lines and/or fibroblast cell lines (transformed into myogenic cells using MyoD transduction; refs. 4,5) derived from the probands of families 12 and 14. Western blot analysis confirmed the absence of LMOD3 expression in myotubes from family 12 and the expression of both mutant forms of LMOD3 in myotubes from patient 14a ( Figure 2B).…”

Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle

“…This remarkable and unexpected fate change was at least one order of magnitude greater than that observed in all previous reported data even in comparison with different types of 'bona fide' stem cells. [32][33][34] Moreover, during coculturing, reversine-treated fibroblasts expressed striated muscle-specific genes such as those encoding MHC and MyoD, before fusion into multinucleated myotubes (Figure 3c-d). Thus myogenesis can be influenced in primary mononucleated fibroblasts by signals emanating from neighboring cells.…”