The ex vivo approach to gene therapy of primary myopathies was designed to overcome at least some of the immunological problems linked to myoblast transplantation. In this strategy, cells are isolated from a muscle biopsy, expanded in vitro, transduced with an appropriate vector encoding the therapeutic gene, and finally reinjected into one or more muscles of the patient from which they had been initially isolated (12). This approach, however, faces two additional problems: first, the difficulty of producing an appropriate, integrating vector to accommodate the very large dystrophin or utrophin cDNAs; and second, the limited life-span of myogenic cells isolated from dystrophic patients. The proliferative potential of human myogenic precursors declines considerably during early postnatal growth, in parallel with the progressive reduction in telomere length which occurs in the first 2 decades of life (13). Given the extra number of cell divisions that myogenic cells from DMD patients undergo in vivo during the various cycles of fiber degeneration and regeneration, their replication capacity is dramatically decreased during early childhood and continues to drop during the first decade of life. These cells are recovered in low number from muscle biopsies, grow poorly in vitro, and rapidly undergo senescence, even though they can be transduced by retroviral vectors with an efficiency comparable to that of normal cells (14, 15). This makes it very difficult to obtain reasonable numbers of genetically modified cells ex vivo and consequently lowers the expectations that this type of strategy might become practical. Attempts to solve the problem of the limited life-span have relied on either immortalization of myogenic cells or recruitment of nonmyogenic cells with a higher proliferative potential. Immortalization of mammalian cells by oncogenes has been successful for decades, and in the case of molecules such as the large T antigen of SV40 or polyoma virus, immortalization is still compatible with a certain degree of differentiation into mature muscle fibers (16, 17). Nevertheless, safety concerns related to the persistence of an active oncogene in the genome of the immortalized cells have precluded, so far, any clinical application of this strategy. In order to overcome these concerns, we designed a retroviral vector expressing the wild-type simian vacuolating virus 40 (SV40) large T antigen and harboring two lox sites in the long terminal repeats. Human primary myogenic cells transduced with this vector showed extended life-spans and retained their differentiation capacity. Transient expression of Cre recombinase allowed the entire provirus to be excised in> 90% of transduced cells, which then underwent terminal differentiation in vivo with an efficiency comparable to that of untreated, primary myogenic cells (18). Reversible immortalization thus allows primary myoblasts to be expanded in culture without compromising their ability to differentiate in vivo and could represent a safe means to increase the availability of these cells for clinical application. Alternative strategies might be conceived along similar lines, such as expressing active telomerase or inhibiting the expression of anti-oncogenes.

To increase the availability of primary myoblasts, we and others have also explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, autologous cells such as skin fibroblasts, the growth potential of which is uncompromised in all primary myopathies. Fibroblasts can be obtained from a number of accessible sources (eg, skin), easily expanded, and genetically modified in culture. If transduced at high efficiency with MyoD, for instance by transient …