MyoDifferentiation

We have previously developed the MyoD-system, an in vitro muscle differentiation system as a way to bypass the lack of muscle tissue or muscle cell cultures from DMD patients. We have initially applied this method for (prenatal) diagnoses of DMD [Roest et al., 1996, 1999]. It is based on the adenoviral delivery of the MyoD gene [the Ad50MyoD vector; Havenga et al., 2002] to patient-derived fibroblasts, amniocytes or chorionic villi cells. Expression of the introduced MyoD gene induces differentiation of the cells into myogenic cells which, within a few days, start to express early muscle proteins like desmin. Through serum deprivation, the cells further differentiate and fuse into multi-nucleated myotubes. A time-dependent range of muscle-specific proteins becomes expressed, including  dystrophin (see Figures below).

We recently also applied this method to test therapeutic exon skipping for DMD patients with specific mutations and from which only fibroblast cultures were available (Aartsma-Rus 2003, 2004, 2007). MyoDifferentiated myotubes expressing relatively high levels of dystrophin were transfected with specific AONs. RNA was isolated at 24 to 48 hours post-treatment and analysed by RT-PCR for appropriate exon skipping. Treated myotubes were also analysed by immunohistochemical analysis to detect de novo dystrophin expression at the membrane.

For future clinical studies on therapeutic exon skipping it will be necessary to pre-screen DMD patients for their suitability and response to AON treatment on RNA level. If no muscle cell cultures are available, a skin biopsy will be taken from which fibroblast cultures can be isolated for MyoDifferentiation.