Retroviral Vector-Mediated Gene Transfer into Human Primary Myogenic Cells Leads to Expression in Muscle Fibers In Vivo

Primary human myogenic cells isolated from fetal and adult muscle were infected with a high-titer, Moloney murine leukemia virus (MoMLV)-derived retroviral vector expressing a bacterial β-galactosidase (β-gal) gene under long terminal repeat (LTR) control. Gene transfer efficiency averaged 50% in both fetal myoblasts and adult satellite cells, as revealed by β-gal staining. The reporter gene was stably integrated, faithfully inherited, and expressed at significant levels in myogenic cells for at least 10 generations under clonal growth conditions, and throughout the culture life span upon differentiation into myotubes. Comparable gene transfer efficiency was obtained in myogenic cells from muscle biopsies of patients affected by a number of genetic or acquired myopathies, including Duchenne muscular dystrophy. Transduced normal human satellite cells were injected into regenerating muscle of immunodeficient mice, where they formed new muscle fibers in which the product of the reporter gene was detectable for 2 months after injection. These results show that retroviral vectors can be used to transfer foreign genes with high efficiency into normal or abnormal primary human myogenic cells, leading to stable expression into mature muscle. Satellite cells engineered in this way might represent an effective tool for gene therapy of muscular dystrophies as well as for systemic delivery of recombinant gene products for correction of inherited and acquired disorders. The human-mouse model described here will allow in vivo testing of such gene therapy approaches. Muscle cells are a key target for development of gene therapy strategies for muscle disorders, as well as for other inherited or acquired diseases through systemic delivery of recombinant proteins by engineered muscle tissue. This study shows that human primary myogenic cells (fetal myoblasts and adult satellite cells) can be transduced with high efficiency by retroviral vectors, and express a reporter β-galactosidase transgene for many cell generations and after differentiation into mature muscle fibers. Transduced satellite cells can be transplanted into immunodeficient mice, where they form striated muscle fibers that retain β-gal activity for at least 2 months. This hybrid human–mouse model will allow direct in vivo testing of the function of genetically modified muscle fibers, and therefore preclinical evaluation of a number of gene therapy strategies based on muscle tissue.