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Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature ( T m ) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.

Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach for subsets of Duchenne muscular dystrophy (DMD) patients to ameliorate the severe DMD phenotype. Several groups have successfully induced exon skipping by AONs to reframe the mRNA in various patients carrying deletions, and phase I/II clinical trials are ongoing. The approach is based on targeting specific splicing motifs, both exonic and located on the exon borders, thus interfering with the spliceosome assembly by steric hindrance. Evaluation of the effectiveness of treatment with AONs in cells, animal models, and humans requires a sensitive, specific, and highly reproducible method. We have developed a real-time PCR-based protocol that uses the probe-based approach to recognize specific sequences internal to the target exon (exon-specific real-time assay). The methods for this protocol are described in this chapter.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

Mutations in the dystrophin gene give rise to Duchenne and Becker muscular dystrophies (DMD and BMD), in which both skeletal and cardiac muscles are affected, but also to X-linked dilated cardiomyopathy (XLDC), a condition characterised by exclusive cardiac involvement. XLDC patients with mutations at the 5' end of the gene typically have a cardiac specific severe transcriptional pathology, with absent dystrophin in the heart, while reduced levels of virtually normal dystrophin transcript and protein are present in the skeletal muscle. We now report the identification of a new XLDC family and the detailed characterisation of the levels of dystrophin protein present in skeletal muscle of this family, and of three previously studied XLDC families. We found that dystrophin levels comprised between 29% and 57% were sufficient to avoid muscle weakness in these XLDC families. This information will be of help for the development of therapeutic approaches aimed at restoring dystrophin levels sufficient to prevent the muscle pathology in DMD.