P.11.21 Bioanalysis of a double blind, placebo-controlled clinical phase 2 study of drisapersen for the treatment of boys suffering from Duchenne muscular dystrophy and comparison to clinical outcome results

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder, ultimately lethal, caused by the absence of dystrophin protein due to mutations of the dystrophin gene. Drisapersen is a 2′-O-methyl-phosphothioate oligo designed to skip exon 51 in the dystrophin pre-mRNA for the treatment of DMD. Here we report the pharmacodynamic results of a double blind, placebo-controlled clinical study of drisapersen. Subjects with DMD and a dystrophin mutation correctable by exon 51 skipping were randomized to 2 drisapersen dosing regimens or matched placebo (2:1) and dosed subcutaneously for 48 weeks. Muscle biopsies were obtained from either the anterior tibialis or quadriceps prior to treatment and at 24 weeks after first dose. The restoration of dystrophin at the sarcolemma was studied by immunofluorescence and semi-automated image analysis. Precision cut cryosections of the muscle biopsies before and after treatment were stained with antibodies against dystrophin and spectrin protein and the resulting images were computationally analysed using customised software to measure sarcolemmal dystrophin intensity in individual fibres. Exon 51 skip induced by drisapersen and the expression of dystrophin in muscle homogenate was evaluated by qualitative RT-PCR and Western blot analysis (WBA), respectively Drisapersen levels were measured using quantitative ELISA methodology, in plasma and muscle tissue. Using immunofluorescence, semi-automated analysis and defined criteria, it was possible to detect changes in dystrophin membrane intensity (mean and quantile values) in the muscle fibre population after treatment with drisapersen. Drisapersen was also observed to induce skip of exon 51 by RT-PCR and dystrophin expression by WBA. This pharmacodynamic data was compared to the primary clinical outcomes including the 6 Minute Walk Distance (6MWD) over 24 weeks, and secondary outcomes included 6MWD at 48 weeks and the North Star Ambulatory Assessment.