Exon-skipping via synthetic antisense oligonucleotides represents one of the most promising potential therapies for Duchenne muscular dystrophy (DMD), yet this approach is highly sequence-specific and thus each oligonucleotide is of benefit to only a subset of patients. The discovery that dystrophin mRNA is subject to translational suppression by the microRNA miR31, and that miR31 is elevated in the muscle of DMD patients, raises the possibility that the same oligonucleotide chemistries employed for exon skipping could be directed toward relieving this translational block. This approach would act synergistically with exon skipping where possible, but by targeting the 3’UTR it would further be of benefit to the many DMD patients who express low levels of in-frame transcript. We here present investigations into the feasibility of combining exon skipping with several different strategies for miR31-modulation, using both in vitro models and the mdx mouse (the classical animal model of DMD), and monitoring effects on dystrophin at the transcriptional and translational level. We show that despite promising results from our cell culture model, our in vivo data failed to demonstrate similarly reproducible enhancement of dystrophin translation, suggesting that miR31-modulation may not be practical under current oligonucleotide approaches. Possible explanations for this disappointing outcome are discussed, along with suggestions for future investigations.
The coordinated differentiation of myoblasts to mature muscle is essential for muscle development and repair, and study of the myogenic program in health and disease is critical to the understanding and treatment of muscle pathologies. Use of quantitative RT-PCR to analyse gene expression in cell culture models of muscle differentiation can be highly informative, but data must be normalized to one or more suitable reference genes. Myogenesis is highly dynamic, thus identification of genes with stable expression throughout this process is challenging. Establishing a common set of reference genes suitable for measuring expression in both healthy and disease models would be of considerable advantage. We measured expression of 11 candidate normalization genes (Cdc40, Htatsf1, Ap3d1, Csnk2a2, Fbxw2, Fbxo38, Pak1ip1, Zfp91, GAPDH, ActB, 18S) in three cell culture models of myogenesis (C2C12 , H2K2B4, and the dystrophic line H2KSF1). Strong and weak normalization candidates were identified using the software packages Bestkeeper, geNorm and Normfinder, then validated against several known myogenic markers (MyoD, myogenin, MEF2C, dystrophin). Our data show that Csnk2a2 and Ap3d1 are suitable for normalizing gene expression during differentiation in both healthy and dystrophic cell-culture models, and that the commonly-used reference standards 18S, ActB and GAPDH are exceptionally poor candidates.
The Sleeping beauty (SB) system is a non-viral DNA based vector that has been used to stably integrate therapeutic genes into disease models. Here we report the SB system is capable of stably integrating the ΔR4-R23/CTΔ micro-dystrophin gene into a conditionally immortal dystrophin deficient muscle cell-line, H2K SF1, a murine cell model for Duchenne muscular dystrophy. Genetically corrected H2K SF1 cells retained their myogenic properties in vitro. Moreover, upon transplantation ΔR4-R23/CTΔ micro-dystrophin expression was detected within mdx nu/nu mice. Our data suggests the SB system is an effective way of stably integrating therapeutic genes into myogenic cells.