Investigating utrophin expression in vivo and in vitro by bioluminescence

Abstract

Duchenne Muscular Dystrophy (DMD) is a lethal muscle-wasting disorder caused by mutations in the DMD gene that results in loss of function of the protein dystrophin. Despite intensive clinical management, there is currently no effective treatment for patients with DMD. One promising therapeutic strategy for treating this disease is to compensate for a lack of dystrophin by enhancing the expression of utrophin, a paralogue that in preclinical studies has been shown to partially rescue dystrophin-deficiency in murine models. Here, I describe the characterisation of novel luciferase-based mouse reporters and myoblast cell lines, in which Dmd and Utrn gene expression can be visualised using bioluminescence. These were generated by inserting genes that encoded spectrally-distinct luciferase proteins, non-disruptively into the 3’ region of endogenous Utrn and Dmd loci, thereby enabling the simultaneous monitoring of both genes in vivo. In order to generate an in vitro model to screen for drugs that increased Utrn expression in adult muscle, I derived a myoblast cell line from Utrn-luciferase reporter mice. Using this approach, I showed that chromatin-modifying drugs can be used to enhance Utrn expression; in a preliminary screen, I discovered that inhibitors of EZH2, the catalytic subunit of the Polycomb Repressive Complex 2 responsible for H3K27 methylation, increase Utrn expression in cultured myoblasts. This result was independently confirmed by genetic ablation of EZH2, which also led to increased Utrn expression in myoblasts. My results also indicated that inhibition of ERK1/2 resulted in an additional increase in Utrn gene expression, opening the possibility of using combinatorial treatments to enhance Utrn expression. Collectively these results show that the luciferase-based mouse and cell lines developed in this work can be used as a platform to examine drugs able to increase Utrn expression that, together with EZH2 and ERK1/2 inhibitors, could provide new therapeutic avenues to explore for the treatment of DMD.