Preclinical PK and PD Studies on 2′-O-Methyl-phosphorothioate RNA Antisense Oligonucleotides in the mdx Mouse Model

Abstract

Antisense oligonucleotides (AONs) are being developed as RNA therapeutic molecules for Duchenne muscular dystrophy. For oligonucleotides with the 2′-O-methyl-phosphorothioate (2OMePS) RNA chemistry, proof of concept has been obtained in patient-specific muscle cell cultures, the mouse and dog disease models, and recently by local administration in Duchenne patients. To further explore the pharmacokinetic (PK)/pharmacodynamic (PD) properties of this chemical class of oligonucleotides, we performed a series of preclinical studies in mice. The results demonstrate that the levels of oligonucleotides in dystrophin-deficient muscle fibers are much higher than in healthy fibers, leading to higher exon-skipping levels. Oligonucleotide levels and half-life differed for specific muscle groups, with heart muscle showing the lowest levels but longest half-life (~46 days). Intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) delivery methods were directly compared. For each method, exon-skipping and novel dystrophin expression were observed in all muscles, including arrector pili smooth muscle in skin biopsies. After i.v. administration, the oligonucleotide peak levels in plasma, liver, and kidney were higher than after s.c. or i.p. injections. However, as the bioavailability was similar, and the levels of oligonucleotide, exon-skipping, and dystrophin steadily accumulated overtime after s.c. administration, we selected this patient-convenient delivery method for future clinical study protocols.

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy

“…The exon skipping levels observed after intramuscular injection with either 2OMePS or PMO AONs were lower than previously observed in the mdx mouse and in cell cultures (Heemskerk et al, 2009a). A pilot experiment with systemic (intravenous) injection of 2OMePS AONs targeting exon 51 in the hDMD mouse resulted in very low or undetectable exon skipping in the muscles (Heemskerk et al, 2010). Recently, vivo-morpholinos against exon 50 were shown to be able to achieve high levels of exon skipping after systemic (intravenous) injection in the healthy skeletal muscles of the hDMD mouse and even low levels in the cardiac muscle.…”

“…After subcutaneous treatment also a decrease in serum creatine kinase (CK) levels was observed. Creatine kinase is an enzyme that leaks out of the muscles into the blood stream when muscles are damaged, so a decrease indicates an improvement of muscle integrity (Heemskerk et al, 2010).…”

Duchenne Muscular Dystrophy: Therapeutic Approaches to Restore Dystrophin

“…However, due to the dystrophic pathology of skeletal muscle in DMD patients, AON uptake is up to 10-fold higher, resulting in sufficient AON levels for exon skipping and dystrophin restoration in skeletal muscles in mdx mice after systemic AON administration (Heemskerk et al, 2009;Lu et al, 2005). In a comparison of intravenous, subcutaneous and intraperitoneal delivery, subcutaneous and intraperitoneal delivery showed the most preferable pharmacokinetic and pharmacodynamic profiles (lower uptake by liver and kidney), while slightly higher exon skipping levels were achieved by intravenous injections (Heemskerk et al, 2010). Based on these results and the relative easy and low invasiveness of subcutenaous injection, this delivery route was selected for systemic clinical trials.…”

“…or microbubbles to improve uptake in heart were used (Alter et al, 2009;Wu et al, 2010). It has become clear that repeated low dose injections are more effective than single high dosage injections (Malerba et al, 2011b;Malerba et al, 2009), probably because of the fast PMOs clearance from the body by the kidneys (Heemskerk et al, 2010). Survival studies show that high doses of PMOs could correct the pathology and were well tolerated (Wu et al, 2011b).…”

Exon Skipping and Myoblast Transplantation: Single or Combined Potential Options for Treatment of Duchenne Muscular Dystrophy

“…In studies in the mdx mouse model, oligonucleotides with chemical properties, similar to the ones of 2'-O-methyl-phosphorothioate (2'OMePS) RNA, were taken up in dystrophin-deficient muscle up to 10 times as much as in healthy muscle tissue, most likely owing to increased permeability of the muscle myofiber membrane. In addition, 4 to 8 weeks' subcutaneous delivery of the oligonucleotides resulted in a steady increase in oligonucleotides levels, exon skipping and dystrophin levels (Heemskerk et al, 2010).…”