A Retroviral Vector Containing a Muscle-Specific Enhancer Drives Gene Expression Only in Differentiated Muscle Fibers

Abstract

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial β-galactosidase (β-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. β-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue. Regulation of retroviral vector-mediated gene expression is a major issue in many gene transfer systems in which tissue-and/or differentiation-specific expression is required. This study reports the development of a retroviral vector containing a muscle-specific enhancer that changes the transcriptional specificity of the viral long terminal repeat (LTR). In transduced muscle progenitors, expression of the transgene is activated only after differentiation into myotubes in vitro or muscle fibers in vivo. Targeted delivery of proteins in the muscle tissue, without accumulation in mononucleated cells, might be relevant for gene therapy approaches in muscular dystrophies based on the use of genetically modified satellite cells.