T.P.2.09 Induced exon skipping in normal and mdx muscle

Induced exon skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce exon skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin exon 23 skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing exon 23 in normal skeletal muscle, as detected by RT-PCR. However, exon 23 skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.