T.P.1 04 Towards safe and efficient full-body delivery of antisense oligonucleotides for the treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is caused by frame-shifting mutations in the DMD gene that lead to dysfunctional truncated dystrophin proteins. By inducing the specific skipping of exons from the pre-mRNA, the reading frame and thus the dystrophin expression can be restored. The resulting proteins carry an internal deletion, but include the essential C-terminal parts, comparable to the partly-functional dystrophins found in the mostly milder Becker muscular dystrophy patients. Exon skipping can be induced with antisense oligonucleotides (AONs) targeting sequences involved in the recognition of exons by the splicing machinery. We have shown that this strategy has therapeutic potential in several patient cell cultures and in the mdx mouse model after intramuscular injections. Our current focus is to find the best AON formulation, dose, regimen, and administration method to deliver AONs safely and efficiently to muscle cells throughout the body. In this study, AON (2′-O methyl PS RNA) uptake in mdx and wild type mice was compared. In addition, different injection sites (intravenous, subcutaneous and intraperitoneal) were evaluated. An AON-specific ELISA showed that AON uptake by muscle fibres was approximately four times higher in mdx mice than in wild type mice, while uptake was equal in other organs. This implies that the damage of the muscle fibre membrane in mdx mice indeed enhances AON uptake. It was further shown that the uptake was highest after IV injection, followed by SC injection and finally IP injection. The levels of AON uptake corresponded to the levels of exon skipping as determined by RT-PCR analysis; the highest skipping levels were observed after IV injection (over 40% in various skeletal muscle groups, 30% in the diaphragm and 5% in the heart). This resulted in significant amounts of dystrophin, even in the heart. Serum creatine kinase levels were decreased when compared to placebo treated littermates, and muscle endurance experiments with a RotaRod measuring device showed a clear improvement.