DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.219 Antisense oligonucleotide-mediated knockdown of IGFBP3 to increase IGF-1 signalling in dystrophic muscle

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by the absence of dystrophin protein. Upon exhaustion of the regenerative capacity of muscle, there is an imbalance between muscle damage and repair. Insulin-like growth factor 1 (IGF-1) plays an essential role in increasing muscle mass and strength, reducing degeneration, inhibiting chronic inflammation and increasing the proliferation potential of satellite cells. Although liver is the main source of IGF-1, it is also expressed in different tissues including skeletal muscle. Both in circulation and the extracellular matrix, IGF-1 is bound to one of its binding proteins (IGFBPs). The majority of IGF-1 transport is associated with IGFBP3. Other IGFBPs are also found in the circulation and complexes of these IGFBPs with IGF-1 can exit the circulation easily, making them more available to target tissues. By contrast, IGF-1+IGFBP3 complexes, unless cleaved by specific proteases, cannot cross vascular endothelium. IGFBP3 is thought to act by inhibiting the availability of IGF-1 to its receptor, which initiates most of the actions of IGF-1. More recently, studies revealed that IGFBP-3 can be inhibitory to myoblast proliferation even in the absence of IGF signalling. Therefore, decreasing IGFBP3 levels would increase IGF-1 action and prevent IGF-1 independent antiproliferative effect of IGFBP3, making it a potential therapy for DMD by improving muscle repair and growth. This can be achieved by downregulation of IGFBP3 using antisense oligonucleotides (AONs) that skip out of frame exon(s), thus preventing the expression of functional protein. In this study, two different AONs were designed to target exon 2 of Igfbp3 and transfected using lipofectamine in mouse C2C12 cell cultures. Cells were harvested after 24 hours and total RNA was isolated. Exon skipping was confirmed by RT-PCR on RNA level. In addition, time course and western blot experiments are ongoing. In vivo studies are being planned.