Exon skipping-mediated downregulation of IGFBP3 protein in mouse C2C12 cells

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by the absence of dystrophin protein. There is an imbalance between muscle damage and repair due to the impaired regenerative capacity of the muscle. Insulin-like growth factor 1 (IGF-1) plays an essential role in muscle maintenance. Other than liver, IGF-1 is also expressed in skeletal muscle. Both in circulation and extracellular matrix (ECM), IGF-1 is bound to binding proteins (IGFBPs). The majority of IGF-1 is bound by IGFBP3 while other IGFBPs are also found in the circulation and ECM. These binding proteins are thought to act by inhibiting the availability of IGF-1 to its receptor, which initiates most of the actions of IGF-1. Therefore, decreasing IGFBP levels would enhance IGF-1 signaling, making it a potential therapy for the treatment of secondary defects in DMD. This can be achieved by antisense oligonucleotides (AONs) that skip out of frame exon(s), thus preventing the expression of functional proteins. In this study, two different AONs were designed to target exon 2 of both Igfbp1 and Igfbp3. The AONs were tested and optimized in C2C12 cells using lipofectamine-mediated transfection with different concentrations. To confirm exon skipping, total RNA was analyzed at 24 h post transfection and RT-PCR was performed. At least one AON was found to be effective for each gene. With these candidate AONs, transfection was repeated to assess the effect of exon skipping on protein level as well. After 48 h, proteins were isolated and downregulation of binding proteins and its effect on the downstream proteins, which are involved in IGF-1 signaling cascade, were confirmed by Western blot. Using exon skipping mediated knockdown of IGFBP1 and IGFBP3, we confirmed in vitro that IGF-1 signaling can be enhanced, making it a potential therapy to be tested in vivo.