T.O.3 Tricyclo-DNA: A promising chemistry for the synthesis of antisense molecules for splice-switching approaches in DMD

Abstract

Abstract Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression in vitro and in vivo. This approach has now been tested in clinical trials with two different chemistries, the 2′O-Methyl phosphorothioate oligoribonucelotides (2′OMe) and the phosphorodiamidate morpholino oligomers (PMO) and has demonstrated encouraging results. However, AO-mediated exon-skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, no crossing of the blood brain barrier, poor cellular uptake and relative rapid clearance from circulation. To overcome these limitations, we investigated the therapeutic potential of a new class of conformationally constrained DNA analogues: the tricyclo-DNAs (Tc-DNA). In this study, we demonstrate widespread restoration of dystrophin in the mdx mouse following systemic injections of Tc-DNA targeting the donor splice site of the dystrophin exon 23. Remarkably, this treatment also leads to restoration of dystrophin in the cardiac muscle and detection of exon skipping in the brain. Moreover, we also show the therapeutic benefit of Tc-DNA treatment in the severely affected utrophin/dystrophin double-knockout mouse (dKO) which is a much more severe and progressive mouse model of DMD. Tc-DNA injections result in significant improvement of their muscle function and dystrophic pathology, by preventing rachiokyphosis and contractures and improving their motility, as assessed by activity monitoring. These findings indicate that Tc-DNA exhibit therapeutic, drug-like effects not only in relatively benign mdx mouse model but also in very severe DMD phenotype of dKO mouse, suggesting great potential for these compounds in the systemic treatment of DMD.